Synthetic methylotrophy to liquid fuels and chemicals

ABSTRACT

A non-naturally occurring microbe capable of growing in a medium comprising methanol is provided. The methanol contributes to a significant percentage (e.g., at least 40%) of the carbon source for the non-naturally occurring microbe, which expresses heterologous methanol dehydrogenase (MDH) and heterologous ribulose monophosphate (RuMP) pathway enzymes. Methods for producing liquid fuels and chemicals by the non-naturally occurring microbe and methods for preparing the non-naturally occurring microbe are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. national phase application of International Application No. PCT/US2015/010795, filed Jan. 9, 2015, claiming the benefit of U.S. Provisional Application No. 61/928,052, filed Jan. 16, 2014, U.S. Provisional Application No. 61/979,058, filed Apr. 14, 2014, U.S. Provisional Application No. 62/023,208, Jul. 11, 2014, U.S. Provisional Application No. 62/061,731, filed Oct. 9, 2014, and U.S. Provisional Application No. 62/091,799, filed Dec. 15, 2014, the contents of each of which are incorporated herein by reference in their entireties for all purposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from the U.S. Advanced Research Projects Agency-Energy (ARPA-E) of Department of Energy (DOE) (Award No. DE-AR0000432). The United States has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to non-naturally occurring microbes expressing heterologous genes required for growing in a medium comprising methanol, and the uses thereof for producing desirable liquid fuels and chemicals.

BACKGROUND OF THE INVENTION

Natural gas consists primarily of methane (CH₄), and includes smaller amounts of higher alkanes, CO₂, N₂, and H₂S. It is used not only for heating and energy generation, but also as a chemical feedstock to produce commodity chemicals that can be then converted to plastics and specialty chemicals. Natural gas constitutes an enormous energy and chemical resource for the US where the recoverable amount is estimated to be 2,000 trillion ft³. Natural gas is however a poor transportation fuel because of its inherently low energy density. Technologies that can convert natural gas into liquid fuels at competitive prices will not only lessen our dependence on imported oil, but also eliminate the needs for retrofitting existing transportation infrastructure. Current chemical routes based on chemical conversion to syngas (CO & H₂) through the Fischer-Tropsch process are not competitive for producing liquid fuels, as they suffer from both high capital costs and low conversion efficiencies. Bioconversion is a promising alternative because of its high specificity and high process energy efficiency all under very mild conditions. Thus, CH₄ represents an ideal target for conversion to liquid fuels by biological processes or hybrid biological/catalytic processes.

While there has been some progresses made in the catalytic conversion of CH₄ to methanol (MeOH), more biological means may be developed for converting methane to methanol, likely by a form of reverse methanogenesis. There remains a need for non-naturally occurring methylotrophic microbes capable of converting methanol efficiently to liquid fuel molecules or other commodity chemicals.

SUMMARY OF THE INVENTION

The present invention relates to non-naturally occurring methylotrophic microbes and methods for use or preparation thereof.

A non-naturally occurring microbe capable of growing in a medium comprising methanol is provided. The methanol contributes to at least 40% of the carbon source for the non-naturally occurring microbe. The non-naturally occurring microbe expresses heterologous methanol dehydrogenase (MDH) and one or more heterologous ribulose monophosophate (RuMP) pathway enzymes. The expression of the one or more RuMP pathway enzymes may be under the control of a formaldehyde responsive promoter. The non-naturally occurring microbe may express heterologous MDH, heterologous 3-hexulose-6-phosphate synthase (HPS), and heterologous 3-hexulose-6-phosphate isomerase (PHI). The non-naturally occurring microbe may contain a deletion of a frmRAB operon.

The non-naturally occurring microbe may further express one or more heterologous pentose-phosphate pathway (PPP) enzymes. The expression of the one or more heterologous PPP enzymes may be under the control of a formaldehyde responsive promoter. The non-naturally occurring microbe may express heterologous phosphofructokinase (PFK), heterologous fructose bisphosphate aldolase (FBA), heterologous transketolase (TKT), heterologous fructose/sedoheptulose biphosphatase (GLPX), heterologous transaldolase (TAL), heterologous ribose-5-phospate (RPI) and heterologous ribulose phosphate epimerase (RPE).

The non-naturally occurring microbe may further express one or more heterologous cyclic formaldehyde dissimilation enzymes. The expression of the one or more heterologous cyclic formaldehyde dissimilation enzymes may be under the control of a formaldehyde responsive promoter. The non-naturally occurring microbe may express heterologous glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND). The non-naturally occurring microbe may contain a deletion of a phosphogluconate dehydratase gene (edd).

The non-naturally occurring microbe may further express one or more heterologous CO₂ fixation pathway enzymes. The expression of the one or more heterologous CO₂ fixation pathway enzymes may be under the control of a formaldehyde responsive promoter. The non-naturally occurring microbe may express heterologous carbonic anhydrase (CA), heterologous formate dehydrogenase (FDH), and heterologous formaldehyde dehydrogenase (FLD).

The non-naturally occurring microbe may further express heterologous dihydroxyacetone synthase (DHAS) and heterologous dihydroxyacetone kinase (DAK).

The non-naturally occurring microbe may be derived from a microbe selected from the group consisting of facultative aerobic organisms, facultative anaerobic organisms, and anaerobic organisms. The non-naturally occurring microbe may be derived from a microbe selected from the group consisting of phyla Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, Chlorobi, and Deinococcus-Thermus. The non-naturally occurring microbe may be derived from a microbe selected from the group consisting of Escherichia, Bacillus, Clostridium, Enterobacter, Klebsiella, Enterobacteria, Mannheimia, Pseudomonas, Acinetobacter, Shewanella, Ralstonia, Geobacter, Zymomonas, Acetobacter, Geobacillus, Lactococcus, Streptococcus, Lactobacillus, Corynebacterium, Streptomyces, Propionibacterium, Synechocystis, Synechococcus, Cyanobacteria, Chlorobi, and Deinococcus. The non-naturally occurring microbe is preferably E. coli.

A method for producing a metabolite is also provided. The production method comprises growing a non-naturally occurring microbe of the present invention in a medium comprising methanol. The methanol contributes to at least 40% of the carbon source for the non-naturally occurring microbe.

The metabolite may be selected from the group consisting of 4-carbon chemicals, diacids, 3-carbon chemicals, higher carboxylic acids, alcohols of higher carboxylic acids, and polyhydroxyalkanoates. Preferably, the metabolite is n-butanol. At least 40% of the carbon in the metabolite may be derived from the methanol. The metabolite may be an amino acid or tricarboxylic acid (TCA) intermediate having a carbon at the fourth position derived from the methanol.

The production method may further comprise modifying the heterologous MDH or any one of the one or more heterologous RuMP pathway enzymes such that the production of the metabolite is improved. The production method may further comprise fixing CO₂.

According to the production method, the non-naturally occurring microbe may be grown at a temperature higher than 37° C. The non-naturally occurring microbe may be grown anaerobically.

A method for making a non-naturally occurring microbe capable of growing in a medium comprising methanol is further provided. The methanol contributes to at least 40% of the carbon source for the non-naturally occurring microbe. The preparation method comprises expressing heterologous methanol dehydrogenase (MDH) and one or more heterologous ribulose monophosophate (RuMP) pathway enzymes in a non-methylotrophic microbe. The non-naturally occurring microbe may express the heterologous MDH, heterologous 3-hexulose-6-phosphate synthase (HPS), and heterologous 3-hexulose-6-phosphate isomerase (PHI).

The preparation method may further comprise expressing one or more heterologous pentose-phosphate pathway (PPP) enzymes in the non-methylotrophic microbe. The non-methylotrophic microbe may express heterologous phosphofructokinase (PFK), heterologous fructose bisphosphate aldolase (FBA), heterologous transketolase (TKT), transaldolase (TAL), heterologous fructose/sedoheptulose biphosphatase (GLPX), heterologous ribose-5-phosphate (RPI), and heterologous ribulose phosphate epimerase (RPE).

The preparation method may further comprise expressing one or more heterologous cyclic formaldehyde dissimilation enzymes. The non-methylotrophic microbe may express heterologous glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND).

The preparation method may further comprise expressing one or more heterologous CO₂ fixation pathway enzymes in the non-methylotrophic microbe. The non-methylotrophic microbe may express heterologous carbonic anhydrase (CA), heterologous formate dehydrogenase (FDH), and heterologous formaldehyde dehydrogenase (FLD).

The preparation method may further comprise expressing heterologous dihydroxyacetone synthase (DHAS) and dihydroxyacetone kinase (DAK).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an overview of the invention. Engineered and optimized E. coli can grow on methanol (MeOH), fix CO₂ & produce various chemicals (such as n-butanol (n-BuOH)). We assume MeOH will be generated biologically or non-biologically from CH₄ with the shown electrons needs/outputs.

FIG. 2 illustrates pathways in an engineered E. coli strain that grows on MeOH, fixes CO₂ and produces n-butanol (n-BuOH) or another desirable chemical through a pathway that departs either from pyruvate or acetyl-CoA. Metabolic Engineering modules: 1 for MeOH use; 2 for CO₂ fixation; 3 for production of various oxychemicals (such as n-BuOH) from intermediates such as acetyl-CoA or pyruvate, but also others in the core cellular pathways.

FIG. 3 shows SDS-PAGE of E. coli cell lysates expressing B. stearothermophilus MDH and M. gastri RuMP fusion. Cells were induced overnight with 0.1 mM IPTG in MOPS+ribose. Lane 1: pETM6Ptac Empty. Lane 2: pETM6PtacBst_MDH/Mg_fusion.

FIGS. 4A-B show A) relative abundance of all labeled fragments and B) relative abundance of each labeled mass isotopomer from labeling experiment with ¹³C-Methanol as tracer. Metabolites shown are 3PG—3-phosphoglycerate, Ser—Serine, Gly—Glycine, Ala—Alanine, Glu—Glutamate, Suc—Succinate, Fum—Fumarate, Mal—Malate, Asp—Aspartate.

FIGS. 5A-B show A) relative abundance of all labeled fragments from cultures incubated for 18 h in MOPS supplemented with either 150 mM, 250 mM, or 500 mM methanol as the sole carbon source, and B) HPLC analysis. The metabolites shown are 3PG—3-phosphoglycerate, Gly—Glycine, Ala—Alanine, Suc—Succinate, Fum—Fumarate, Mal—Malate, Glu—Glutamate. The HPLC analysis confirmed that methanol was simultaneously being consumed from the media.

FIGS. 6A-B show E. coli containing either the pETM6_BstMDH_MgRuMP or pETM6_Empty where grown either A) MOPS+500 mM methanol+2 mM glucose. B) Methanol incorporation was detected in these cultures and the relative abundance of fragments is shown. The first bar indicates samples taken after 6 h of growth and the second bar indicates samples taken after 18 h of growth.

FIGS. 7A-B show growth of MeOH utilizing E. coli in mixed methanol and LB media. The MeOH utilizing strain and the empty vector control were pre-cultured and induced overnight in MOPS+ribose. The cells were pelleted and resuspended in 1:4 mixture of LB:MOPS+methanol. This media contains the following nutrient sources: 2.5 g/l tryptone; 1.25 g/l yeast extract; and 500 mM methanol. Change is cell concentration was measured spectrophotometrically and methanol consumption was measured through HPLC analysis.

FIGS. 8A-B show growth of cultures under anaerobic conditions in MOPS+glucose or MOPS+glucose+500 mM methanol. Strains were pre-grown aerobically in LB before being induced overnight aerobically in MOPS+ribose. After induction, the cultures were transferred to MOPS+2 mM Glucose+500 mM methanol in sealed anaerobic bottles and grown for the allotted time. Enhanced growth was observed when methanol and 0.15 mM methyl viologen were added to the cultures.

FIGS. 9A-B show variations of the ribulose monophosphate pathway commonly found in methylotrophic bacteria: A) Entner-Doudoroff Variant and B) Fructose Bisphosphate Variant. MeOH, methanol; HCHO, formaldehyde; F6P, fructose-6-phosphate; FBP, fructose 1,6-bisphosphate; G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; KDPG, 2-keto-3-deoxy-6-phosphogluconate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; X5P, xyulose-5-phosphate; E4P, erythrose-4-phosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose 1,7-bisphosphate; RSP, ribose-5-phosphate; Ru5P, ribulose-5-phosphate;

FIG. 10 shows SDS-PAGE of E. coli cell lysates expressing RuMP assimilatory pathway genes. Cells were induced overnight with 0.1 mM IPTG in MOPS+ribose. Lane 1: pCDM4_Acessory genes. Lane 2: pETM6PtacBst_MDH/Mg_fusion/pCDM4_Accessory genes.

FIG. 11 shows conceptual illustration of a dual plasmid reporter system. A double plasmid containing E. coli strain will house the plasmids, pETM6_Ptac_MDHlib and pCDM4_Pfrm_GFP. Upon MDH library expression with IPTG, MeOH will be added and oxidized to HCHO by active MDH mutants. GFP expression will then be utilized to select desirable MDH mutants via FACS.

FIG. 12 shows response curve for E. coli pETM6_Pfrm_GFP against HCHO. GFP expression correlated with HCHO concentration over time. Overnight LB cultures were transferred to MOPS+0.1% glucose for the experiment. No growth was observed in 2 and 4 mM HCHO cultures, explaining the absence of GFP expression.

FIG. 13 shows response curve for E. coli pACM4_pFRM_GFP against HCHO. GFP expression correlated with HCHO concentration over time. Overnight LB cultures were transferred to MOPS+0.1% glucose for the experiment. Retarded growth was observed in 500 and 1000 μM HCHO cultures, explaining the decreased of GFP expression.

FIG. 14 shows response curve for E. coli pACM4_pFRM_GFP+pETM6-BstMDH against methanol. GFP expression correlated with methanol concentration over time. Overnight LB cultures were transferred to MOPS+0.1% glucose for the experiment. Retarded growth was observed in 2000 and 4000 mM methanol cultures, explaining the decreased of GFP expression.

FIG. 15 shows ¹⁴C incorporation into the cell mass for Candida boindini (CanBo) and the Clostridium carboxidivorans (Cloca) FDH overexpressing E. coli strains.

FIG. 16 shows heterologous co-expression of the Moorella FDH-A and FDH-B subunit in E. coli. The FDH-B overexpression band had the expected size of approximately 75 kDa, FDH-A was expressed only as a truncated protein of 25 kDa for both the Genscript and DNA 2.0 cDNAs.

FIGS. 17A-B show A) time course data and B) specific activities (calculated from 10 minute time points) for in vivo assays using E. coli BL21(DE3) MDH strains and 1M MeOH. One unit (U) is defined as μmol HCHO produced per minute. Error bars represent averages from two biological replicates.

FIGS. 18A-B show A) time course data and B) specific activities (calculated from 10 minute time points) for in vivo assays using E. coli ΔfrmA MDH strains and 0.5M MeOH. One unit (U) is defined as μmol HCHO produced per minute.

FIG. 19 shows specific activities (calculated from 10 minute time points) for in vivo assays using E. coli ΔfrmA MDH strains and either 0.5 M MeOH for MeOH oxidation or 0.5 mM HCHO for HCHO reduction. One unit (U) is defined as μmol HCHO produced or consumed per minute.

FIG. 20 shows specific activities in an in vivo assay at increased temperatures. Data was obtained from E. coli BL21(DE3) pETM6_Bme MGA3 Mdh3 and 1M MeOH. The first temperature represents the growth temperature while the temperature in parentheses represents the temperature at which the assay was performed. One unit (U) is defined as μmol HCHO produced per minute. Error bars represent averages from two biological replicates, except in the case of 40° C. (40° C.), which is from one biological replicate.

FIG. 21 shows specific activities in an in vitro assay demonstrating that all MDHs possess higher activity towards butanol compared to methanol. As presented, the MDHs exhibit a wide range of alcohol selectivity. 100 mM alcohols were used. One unit (U) is defined as μmol NADH produced per minute. Error bars represent averages from two biological replicates.

FIG. 22 shows aldehyde indicator plates. E. coli BL21(DE3) containing Bme MGA3 Mdh3 in pETM6 is represented. The arrow indicates a positive colony producing formaldehyde, which appears red via the reaction with pararosaniline and sodium bisulfite.

FIGS. 23A-B show growth and methanol consumption by two E. coli strains, an empty vector control (pM6 Empty) and a strain expressing the MDH from B. stearothermophilus and the RuMP pathway from L3 (pM6_BsMDH_MgRuMP). Both strains were grown to an OD₆₀₀ of 0.4 in LB broth and induced with 1 mM IPTG for 3 hours. The strains were then pelleted, washed, and resuspended in MOPS media containing 1% MeOH and 0.1% glucose and allowed to grow overnight so that the cells could become adapted to the minimal media. The next day the cells were resuspended in MOPS containing 1% MeOH and 0.04% glucose. Glucose was added to the cultures at a concentration of 0.04% every hour and aliquots were taken every two hours for HPLC analysis (A) and changes in OD₆₀₀ (B).

FIG. 24 shows formaldehyde consumption by the native E. coli strain BW25113, the ΔfrmA mutant which has a nonfunctional formaldehyde detoxification pathway, and the ΔfrmA mutant expressing the HPS and PHI genes of M. gastri (ΔfrmA_MgRUMP fusion). All three strains were grown to mid-log phase (OD₆₀₀ of 0.4). At this time, 1 mM IPTG was added to the cultures and they were allowed to grow for an additional 3 hours. The cultures were then washed with MOPS and resuspended to an OD₆₀₀ of 0.5 in fresh MOPS buffer containing 0.5 mM formaldehyde. Aliquots were taken at 0, 20, 40, 60, and 90 minutes to determine formaldehyde concentration using the NASH reagent.

FIG. 25 shows the pETM6-tac vector which will be used in a cloning strategy. The ePathBrick set of vectors (pCDM4, pACM4, pETM6) were modified by replacing the T7 promoter with the inducible tac promoter while keeping all other functional portions of the ePathBrick vectors, including the isocaudamer cloning sites.

FIG. 26 shows formaldehyde inducible gene expression in E. coli. The pETM6 T7 promoter was replaced by the 200-bp region upstream of the frmRAB operon and assayed to see if this region contained a promoter that responds to increasing formaldehyde. GFP was cloned into the multiple cloning site of the vector. Cultures of this strain were grown in LB to mid-log phase (OD₆₀₀ of 0.4) and were either induced with 0.5 mM formaldehyde or left uninduced. Aliquots were taken immediately prior to induction, 1 hour, and 2 hours post-induction with formaldehyde, and analyzed for GFP expression using a flow cytometer. pUC-GFP served as a positive control for GFP expression (induced with 1 mM IPTG) and a strain containing pETM6_pFrm_empty served as a negative control.

FIGS. 27A-B show A) pACM4 vector and B) pETM6 vector for cloning genes driven by the inducible tac promoter.

FIG. 28 shows the amount of ¹³CO₂ in the headspace after 4 hours of cultures grown with ¹³C-Formaldehyde.

FIG. 29 shows the amount of formaldehyde in media after 4 hours of cultures grown with ¹³C-Formaldehyde.

FIG. 30 shows ¹³C-labeling of metabolites in central carbon metabolism. Suc289—Succinate. Fum287—Fumarate. Ma1419—Malate. PEP453—Phosphoenolpyruvate. 3PG585—3-phosphoglycerate. Asp418—Aspartate. Glu432—Glutamate

FIG. 31 illustrates the expected labeling patterns using labeled formaldehyde. The black circles are labeled carbons and the white circles are unlabeled carbons. Percentages are the relative abundance of M+1 labeled fragments (FIG. 17). The RuMP pathway is represented by the arrows.

FIGS. 32A-C shows A) the final optimized sequence (SEQ ID NO: 1) for higher expression levels of the Moth FDH-A in E. coli with the artificially designed SECIS element designed by DNA2.0, including Selenocysteine codon (bold), Moth FDH-A native sequence (underlined) and E. coli SECIS optimized sequence (bold and italic), B) optimized sequence designed by Genscript (SEQ ID NO: 2), and C) the frmRAB operon (SEQ ID NO: 3) in E. coli BL21 (DE3), including frmR (underlined), frmA (bold), frmB (underlined and italic), upstream gene of frmR (underlined and bold), and the regions adjacent to the frmAB operon (bold and italic) to design primers.

FIG. 33 shows MDH candidates possessing in vivo activity. All nine MDH candidates were assayed in vivo; only those resulting in detectable activity are shown for clarity. pETM6 represents the empty vector control.

FIG. 34 shows specific activities calculated from initial reaction rates of FIG. 33. One unit (U) is defined as the μmol of formaldehyde produced per minute.

FIG. 35 shows protein expression of MDH and ACT in recombinant E. coli strains. Lanes: 1) Marker, 2) pETM6 empty vector, 3) Bst 2334 Mdh, 4) Bst NUB3621 Mdh, 5) Bme MGA3 Mdh2, 6) Bme MGA3 Mdh3, 7) Bst 2334 Mdh+Bst NUB3621 Act, 8) Bst NUB3621 Mdh+Bst NUB3621 Act, 9) Bme MGA3 Mdh2+Bme MGA3 Act, 10) Bme MGA3 Mdh2+Bme MGA3 Act. Bst MDHs are 36.2 kDa, Bme MDHs are 40.7 kDa, and ACTs are 21 kDa.

FIG. 36 shows growth of E. coli in M9 media plus methanol. Induced cells were resuspended in M9 media supplemented with 0.75% methanol, 0.04% ribose and 0.005% fucose. Growth was monitored by optical density.

FIGS. 37A-B show methanol consumption by E. coli BL21(DE) containing either (A) pETM6_Bs_MDH_L3RuMP or (B) pETM6_Empty. HPLC analysis of culture supernatants was used to monitor methanol consumption.

FIG. 38 shows RuMP pathway accessory genes cloned into the pCDM4 vector backbone in the pseudo-operon configuration.

FIGS. 39A-C shows A) Moorela FDH-A sequence with E. coli SECIS (2,682 nt, 894aa) (SEQ ID NO: 8), including Selenocysteine codon (bold), Moorela native sequence (underlined) and E. coli sequence for E. coli SECIS optimization (bold and italic); B) optimized recombinant M. thermoacetica FDH-A (SEQ ID NO: 9), and C) optimized M. thermoacetica FDH-B (SEQ ID NO: 10).

FIG. 40 illustrates the reverse tricarboxylic acid (rTCA) cycle.

FIGS. 41a-c show measurement of methanol dehydrogenase (MDH) activity in vitro of expressed enzymes of B. stearothermophilus and B. methanolicus PB1 along with the effect of an activator protein from B. methanolicus on MDH activity. The assay was adapted from Krog et al. (PLOS One. 2013; 8(3):59188) with the reduction of NAD+ measured at 340 nm. All assays were performed using crude E. coli cell extracts from strains containing the respective genes cloned into pETM6. B. stearothermophilus MDH is not stimulated by B. methanolicus PB1 ACT in any ratio (a). B. methanolicus PB1 MDH2 is stimulated by B. methanolicus PB1 ACT (b). Michaelis-Menten characterization of methanol dehydrogenases (c), where EXP represents experimental data and MM represents corresponding Michaelis-Menten fit.

FIGS. 42A-B show growth of E. coli expressing HPS and PHI genes from M. gastri. The ability of E. coli to grow in A) LB or B) mixed M9:LB (3:1) spiked with formaldehyde was assessed. Log phase cultures of E. coli harboring pET_M.g.PHI_HPS (circles) or pET21_Empty (squares) were induced with 0.5 mM IPTG for 3 h before being diluted into test media containing 2 mM formaldehyde (LB) or 1.5 mM formaldehyde (M9:LB). Aliquots were taken from the cultures at the indicated time points to determine the optical density (OD₆₀₀). Arrows indicate times in which the cultures were spiked with (A) 2 mM or (B) 1.5 mM formaldehyde.

FIG. 43 shows consumption of formaldehyde by E. coli cells expressing the M. gastri phi and hps genes in M9 minimal Media+0.25 mM formaldehyde. Log phase cultures of E. coli harboring pET_M.g.PHI_HPS (circles) or pET21_Empty (squares) were induced with 0.5 mM IPTG for 3 h before being diluted into M9 minimal media containing 0.25 mM formaldehyde as the sole carbon source. Aliquots were taken from the cultures at the indicated time points to determine the optical density (OD₆₀₀). Culture formaldehyde concentrations for pET_M.g.PHI_HPS (open circles; dashed line) and pET_Empty (open squares; dashed line) were determined via the Hantzch reaction. Arrows indicate times in which the cultures were spiked with 0.25 mM formaldehyde.

FIGS. 44A-D show heterologous-expression and enzymatic activity assays for formaldehyde dehydrogenases from C. boindini (CanBo), R. sphaeroides (Rhodo) and M. flagellatus (M8). (A) SDS-PAGE protein gel showing expression levels for GenCanBo (CanBo 1), DNA2.0 (CanBo 2) and Rhodo 1. (B) C. boindini formaldehyde dehydrogenase activity monitoring NADH synthesis at OD340. Assay buffer and PET21A empty vector lysate were tested as negative controls. (C) R. sphaeroides and M. flagellatus formaldehyde dehydrogenase activity monitoring NADH synthesis at OD340. PET21A empty vector lysate was tested as negative control. (D) C. boindini (CanBo) and R. sphaeroides (Rhodo) formaldehyde dehydrogenase activity in reverse direction (NADH consumption for reduction of formate to formaldehyde).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to engineering E. coli or other microbes that do not naturally grow on or metabolize methanol to become methylotrophic, that is, capable of using methanol for growth as a sole substrate or co-substrate together with various carbohydrates or other carbon and energy substrates. The resulting non-naturally occurring microbes are capable of using the reduction energy from methanol utilization to fix CO₂, and produce liquid fuel and chemicals. This technology integrates all critical components required for achieving the overall goal of cost-efficient biofuel production starting from methanol (but ultimately CH₄) while at the same time minimizing CO₂ release.

Methanol (MeOH) utilization (FIG. 1 and Module 1 in FIG. 2) and CO₂ fixation (Module 2 in FIG. 2) may take place simultaneously to produce various chemicals and fuel molecules, for example, n-butanol (n-BuOH; FIGS. 1 & 2) from acetyl co-A or pyruvate or other intermediates of glycolysis, the TCA (tricarboxylic acid) cycle and related core pathways of the cells. The reduction energy contained in MeOH, which is more reduced than certain sugars, e.g., glucose (see reaction 1 of FIG. 2) may be conserved under anaerobic growth conditions and used to produce various oxygenated molecules such as BuOH and at the same time fix CO₂. To achieve this, genes/enzymes for the 3 modules, i.e., MeOH utilization, CO₂ fixation, and product formation (e.g., n-BuOH formation) (FIG. 2), are assembled in a microbe. Each of these goals may be carried out efficiently, and assessed for effectiveness of outcomes by different methods, for example, in terms of rates of MeOH and CO₂ utilization, carbon fluxes to pyruvate or acetyl-CoA, and the engineered pathway to produce the desirable product such as n-BuOH (FIG. 2).

The present invention provides an approach to simultaneously use MeOH and CO₂ to produce n-butanol (n-BuOH) (FIG. 2) or other chemicals starting with acetyl-CoA, pyruvate, or other intermediate metabolites (or chemicals) of the glycolysis pathway or pentose phosphate pathway, including but not limited to D-xylulose 5-phosphate, D-glyceraldehyde 3-phosphate and/or glycerone. This may be achieved by leveraging genes from methylotrophs for MeOH utilization (Module 1), and various natural or synthetic pathways for CO₂ fixation (Module 2) using genes from acetogens and other organisms. Synthetic methylotrophic strains may be engineered by combining these two modules with natural or synthetic pathways to produce a chemical or biofuel, such as for n-BuOH production (Module 3). Exemplary chemicals include 2, 3, 4, 5 and 6 carbon alcohols, carboxylic acids, ketones, aldehydes and di-acids.

Methanol may be used as a carbon source by conversion to formaldehyde (HCHO) by a MeOH dehydrogenase (MDH). HCHO may then be converted to hexulose-6-phosphate, using ribulose-5-phosphate, by a 3-hexulose-6-phosphate synthase (HPS). 3-Hexulose-6-phosphate isomerase (PHI) may convert the hexulose-6-phosphate to fructose-6-phosphate, which may then be used for generation of pyruvate and subsequently acetyl-CoA that may be the starting chemical to feed carbon skeletons into various synthetic pathways to produce various oxychemicals in, for example, the n-BuOH producing pathway (FIG. 2, module 3).

E. coli or the other microbes may be engineered to utilize MeOH as a carbon and energy source by expressing a MeOH dehydrogenase (MDH) and enzymes from the methylotrophic RuMP pathway (e.g., HPS and PHI, FIG. 2). Expression of additional pentose-phosphate pathway (PPP) enzymes as an additional inventive step and strategies including suitable expression promoters may be used to enable or enhance the expression of the corresponding genes in cells when exposed to methanol and/or formaldehyde as substrates.

Significantly, the present invention provides additional steps to make possible the recycling of all or most evolved CO₂ from the decarboxylation of pyruvate to acetyl-CoA (FIG. 2) aiming to minimize CO₂ formation. This can be executed for other biological systems independent of MeOH utilization as long as the cells use a carbon substrate that generates sufficient electrons available for CO₂ fixation and also as long as the cells can incorporate formaldehyde into metabolic intermediates. The engineered methylotrophic E. coli or other microbes may be generated for high pathway energy efficiency, yield, and kinetics.

The terms “protein” and “polypeptide” are used herein interchangeably, and refer to a polymer of amino acid residues with no limitation with respect to the minimum length of the polymer. Preferably, the protein or polypeptide has at least 20 amino acids. The definition includes both full-length proteins and fragments thereof, as well as modifications thereof (e.g., glycosylation, phosphorylation, deletions, additions and substitutions). The protein may be an enzyme involved in a biological pathway.

The term “polynucleotide” used herein refers to a polymer of nucleotide residues with no limitation with respect to the minimum length of the polymer. Preferably, the polynucleotide has at least 60 nucleotides. The polynucleotide may be a DNA, cDNA or RNA molecule. A polynucleotide may comprise a gene encoding a desirable protein (e.g., an enzyme), optionally under the control of an inducible promoter.

The term “variant” of a protein or polynucleotide used herein refers to a polypeptide having an amino acid or nucleic acid sequence that is the same as the amino acid or nucleic acid sequence of the protein or polynucleotide except having at least one amino acid or nucleic acid modified, for example, deleted, inserted, or replaced, respectively. A variant of a protein or polynucleotide may have an amino acid or nucleic acid sequence at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, identical to the corresponding amino acid sequence or nucleic acid of the protein or polynucleotide.

The term “derived from” used herein refers to the origin or source, and may include naturally occurring and recombinant microorganisms or molecules, or variants thereof. For example, a gene derived from a bacteria may be identical to the corresponding native gene or a variant thereof in the bacteria, i.e., having a nucleic acid sequence at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95%, identical to the corresponding native gene.

The present invention provides a non-naturally occurring microbe capable of growing in a medium comprising methanol. The methanol contributes to a significant percentage of the carbon source for the microbe. The microbe expresses heterologous methanol dehydrogenase (MDH) and one or more heterologous ribulose monophosphate (RuMP) pathway enzymes.

The term “a significant percentage of the carbon source” used herein refers to that the methanol contributes to at least about 40%, 48%, 50%, 60%, 66%, 70%, 80%, 90%, 95%, 99%, or 100% of the carbon source for the non-naturally occurring microbe. Preferably, the methanol may contribute to at least about 40% of the carbon source. Methanol could be also the sole carbon source, i.e., contributing 100% of the carbon source, for the non-naturally occurring microbe.

The non-naturally occurring microbe is not naturally a methylotrophic microbe, but with this invention, it becomes a methylotrophic microbe. The term “methylotrophic microbe” used herein refers a microbe capable of growing in a medium comprising methanol, which contributes to as least about 40%, 48%, 50%, 60%, 66%, 70%, 80%, 90%, 95%, 99%, or 100%, preferably at least about 40%, more preferably 100%, of the carbon source for the methylotrophic microbe.

The term “microbe” used herein refers to a single cell organism. Examples of microbes include bacteria, archaea, and fungi.

The non-naturally occurring microbe of the present invention may be derived from a microbe selected from the group consisting of facultative aerobic organisms, facultative anaerobic organisms, and anaerobic organisms. In particular, the non-naturally occurring microbe may be derived from a microbe in phyla Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, Chloribi, and Deinococcus-Thermus. For example, the non-naturally occurring microbe may be derived from Escherichia, Bacillus, Clostridium, Enterobacter, Klebsiella, Enterobacteria, Mannheimia, Pseudomonas, Acinetobacter, Shewanella, Ralstonia, Geobacter, Zymomonas, Acetobacter, Geobacillus, Lactococcus, Streptococcus, Lactobacillus, Corynebacterium, Streptomyces, Propionibacterium, Synechocystis, Synechococcus, Cyanobacteria, Chlorobi, or Deinococcus. Preferably, the non-naturally occurring microbe of the present invention is E. coli.

The heterologous methanol dehydrogenase (MDH) is an enzyme capable of converting methanol to formaldehyde (HCHO) in the non-naturally occurring microbe of the present invention. The heterologous MDH may be derived from any other microbe, for example, Bacillus stearothermophilus or other naturally facultative methylotrophs such as B. methanolicus. The expression of the heterologous MDH may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde responsive promoter, a methanol inducible promoter, a lactose inducible promoter, or a temperature or pH responsive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically.

The term “ribulose monophosphate (RuMP) pathway” as used herein refers to a formaldehyde assimilation pathway in a microbe, which fixes formaldehyde produced via methanol oxidation to the central metabolite ribulose-5-phosphate. Exemplary RuMP pathway enzymes include 3-hexulose-6-phosphate synthase (HPS), and 3-hexulose-6-phosphate isomerase (PHI). The heterologous RuMP pathway enzymes may be derived from any microbe, for example, M, gastri, B. brevis, B. subtilis, B. methanolicus, Methylobacillus flagellatus, or Methylomonas str. L3, or other obligate or facultative aerobic or anaerobic methylotrophs. Preferably, the RuMP pathway enzymes may be derived from the same microbe. The RuMP pathway enzymes may be expressed as a fusion protein. For example, the heterologous HPS and the heterologous PHI may be expressed as a fusion protein. The non-naturally occurring microbe of the present invention may have any native formaldehyde detoxification system such as the frmRAB operon. The non-naturally occurring microbe may further contain a deletion of the frmRAB operon or deletion of a similar set of genes that code for enzymes that oxidize formaldehyde to CO formaldehyde typically for formaldehyde detoxification purposes. The expression of any one of the heterologous RuMP pathway enzymes may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde responsive promoter, a lactose inducible promoter, or temperature sensitive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. In some preferred embodiments, the non-naturally occurring microbe expresses heterologous MDH, heterologous HPS, and heterologous PHI.

The non-naturally occurring microbe of the present invention may further express heterologous pentose-phosphate pathway (PPP) enzymes. The term “pentose-phosphate pathway (PPP)” as used herein refers to a cyclic metabolic pathway which functions to regenerate the ribulose-5-phosphate used by the RuMP pathway. Exemplary PPP enzymes include phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), transketolase (TKT), fructose/sedoheptulose biphosphatase (GLPX), ribulose phosphate epimerase (RPE), ribose-5-phosphate isomerase (RPI) and transaldolase (TAL). The heterologous PPP enzymes (e.g., PFK, FBA, TKT, GLPX, RPE, RPI, and TAL) may be derived from any microbe, for example, any bacterium, archaeon, fungus or even animal cells as long as the genes have been optimized for expression in the host organism as is now well practiced by those skilled in the art. Preferably, the heterologous PPP enzymes are derived from the same microbe. Some or all of the PPP pathway enzymes may be expressed as a fusion protein. The expression of any one of the heterologous PPP enzymes may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde or methanol responsive promoter, a lactose inducible promoter, or a temperature or pH responsive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. In some preferred embodiments, the non-naturally occurring microbe expresses heterologous MDH, heterologous HPS, heterologous PHI, heterologous PFK, heterologous FBA, heterologous TKT, heterologous GLPX, heterologous TAL, heterologous RPI, and heterologous RPE.

The non-naturally occurring microbe of the present invention may further express one or more heterologous cyclic formaldehyde dissimilation enzymes. Exemplary cyclic formaldehyde dissimilation enzymes include glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND). The non-naturally occurring microbe of the present invention may contain a deletion of the phosphogluconate dehydratase gene (EDD). The heterologous cyclic formaldehyde dissimilation enzymes may be derived from any microbe. Some or all of the cyclic formaldehyde dissimilation enzymes may be expressed as a fusion protein. The expression of any one of the heterologous cyclic formaldehyde dissimilation enzymes may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde responsive promoter, a lactose inducible promoter, or temperature sensitive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. In some preferred embodiments, the non-naturally occurring microbe expresses heterologous MDH, heterologous HPS, heterologous PHI, heterologous PFK, heterologous FBA, heterologous TKT, heterologous GLPX, heterologous TAL, heterologous RPI, heterologous RPE, heterologous PGI, heterologous ZWF, heterologous PGL, and heterologous GND.

The non-naturally occurring microbe of the present invention may further express heterologous CO₂ fixation pathway enzymes. The term “CO₂ fixation pathway” as used herein refers to the ability of a microbe to utilize CO2 or it salts such as various mono and bicarbonate salts. Exemplary CO₂ fixation pathway enzymes include carbonic anhydrase (CA), formate dehydrogenase (FDH), formaldehyde dehydrogenase (FLD); the enzymes of the reductive tricarboxylic acid cycle such as ATP citrate lyase (ACL), 2-oxoglutarate: ferredoxin oxidoreductase (OGOR), isocitrate dehydrogenase (ICDH), and fumarate reductase (FR); the enzymes of the glycine cleavage system such as aminomethyltransferase (AMT), dehydrolipoyl dehydrogenase (LPDH), glycine dehydrogenase (GDH); and the enzymes of the non-oxidative glycolysis pathway including fructose phosphoketolase, xylose phosphoketolase, transaldolase, transketolase, fructose 1,2-bisphosphate aldolase, fructose 1,6-bisphosphatase, ribulose-5-phosphate epimerase, ribose-5-phosphate isomerase, and trios phosphate isomerase.

The heterologous CO₂ fixation pathway enzymes such as carbonic anhydrase (CA) (EC 4.2.1.1), formate dehydrogenase (FDH) (EC 1.2.1.43 or EC1.2.1.2) and formaldehyde dehydrogenase (FLD) (EC 1.1.1.284) may be derived from several microbes (or host cells), for example, E. coli, acetogenic bacteria, various yeasts or even animal cells. Some or all of the heterologous CO₂ fixation pathway enzymes may be expressed as a fusion protein. The expression of any one of the heterologous CO₂ fixation pathway enzymes may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde responsive promoter, a lactose inducible promoter, or temperature sensitive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. In some preferred embodiments, the non-naturally occurring microbe expresses heterologous MDH, heterologous HPS, heterologous PHI, heterologous PFK, heterologous FBA, heterologous TKT, heterologous GLPX, heterologous RPE, heterologous RPI, heterologous TAL, heterologous PGI, heterologous ZWF, heterologous PGL, heterologous GND, heterologous CA, heterologous FDH, and heterologous FLD.

The non-naturally occurring microbe of the present invention may further express heterologous dihydroxyacetone synthase (DHAS, EC=2.2.1.3), which is also known as formaldehyde transketolase or glycerone synthase. Additionally, the non-naturally occurring microbe may further express heterologous dihydroxyacetone kinase (DAK, EC=2.7.1.29), which is also known as glycerone kinase. The DHAS and DAK may be derived from any microbe, for example, any methylotrophic yeast or from the bacterium Mycobacterium sp. JCL The expression of any one of the heterologous DHAS and heterologous DAK enzymes may be under the control of a constitutive or an inducible promoter, for example, a formaldehyde responsive promoter, a lactose inducible promoter, or temperature sensitive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically In some preferred embodiments, the non-naturally occurring microbe expresses heterologous MDH, heterologous HPS, heterologous PHI, heterologous PFK, heterologous FBA, heterologous TKT, heterologous GLPX, heterologous TAL, heterologous RPI, heterologous RPE, heterologous PGI, heterologous ZWF, heterologous PGL, heterologous GND heterologous CA, heterologous FDH, heterologous FLD, heterologous DHAS, and heterologous DAK.

The present invention also provides a method for producing a metabolite. The method comprises growing a non-naturally occurring microbe of the present invention in a medium comprising methanol. The methanol contributes to a significant percentage of the carbon source for the non-naturally occurring microbe.

The methanol may contribute to at least about 40%, 48%, 50%, 60%, 66%, 70%, 80%, 90%, 95%, 99%, or 100% of the carbon source for the non-naturally occurring microbe. Preferably, the methanol may contribute to at least about 40% of the carbon source. More preferably, the methanol is the sole carbon source, i.e., contributing 100% of the carbon source, for non-naturally occurring microbe.

The medium may further comprise other carbon source, for example, fermentable mono, di, oligo or polysaccharides. Exemplary fermentable monosaccharides include glucose, xylose, mannose, arabinose, rhamnose, and ribose. Fermentable di- or oligosaccharides may be sucrose, lactose, maltose, cellobiose, short polymers of these mono- or di-saccharides, or long polymers of saccharides, for example, cellulose and xylan. The other carbon source may contribute to no more than about 40%, preferably no more than about 30%, more preferably no more than about 20%, most preferably no more than about 10% of the carbon source for the non-naturally occurring microbe.

The metabolite may be selected from the group consisting of 4-carbon chemicals, diacids, 3-carbon chemicals, higher carboxylic acids, alcohols of higher carboxylic acids, polyhydroxyalkanoates, and specialty chemicals. The 4-carbon chemicals may be selected from the group consisting of butyrate, n-butanol, i-butanol, 2-butanol, 2,3-butanediol, and 1,4-butanediol. The diacids may be selected from the group consisting of oxalic, malonic, succinic, glutaric, adipic, pimelic, pthalic, isopthalic, and terephtlalic. The 3-carbon chemicals may be selected from the group consisting of propanol, propanediol, lactate, and acrylate. The higher carboxylic acids may be selected from the group consisting of pentanoic acids and hexanoic acids. Preferably, the metabolite is n-butanol. The specialty chemicals may include artemisinin, vanillin, anthocyanins, resveratrol, et cetera.

According to the method of the present invention, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, preferably at least about 80%, of the carbon in the metabolite is derived from the methanol. In some embodiments, the metabolite is an amino acid or tricarboxylic acid (TCA) intermediate having at one or multiple carbon positions of the chemical up to the fourth position derived from the methanol. The present method may produce a desirable metabolite at least about 100 mg/L(iter) for specialty, high-value chemicals, and at least 1 g/L or higher for commodity, less expensive chemicals and biofuel molecules.

The growing conditions for the non-naturally occurring microbe may be modified to improve the metabolite production or methanol utilization. For example, the non-naturally occurring microbe may be grown anaerobically, or at a temperature higher than 37° C., for example, 40° C., 45° C. or 50° C.

A gene encoding a heterologous enzyme, for example, MDH, the RuMP pathway enzymes (e.g., HPS and PHI), the PPP pathway enzymes (e.g., PFK, FBA, TKT, TAL, GLPX, RPI, and RPE), the cyclic formaldehyde dissimilation enzymes (e.g., PGI, ZWF, PGL, and GND), the CO₂ fixation pathway enzymes (e.g., CA, FDH, FLD, reductive tricarboxylic acid cycle enzymes such as ACL, OGOR, ICDH, and FR, glycine cleavage system enzymes such as AMT, LPDH, GDH, non-oxidative glycolysis pathway enzymes such as fructose phosphoketolase, xylose phosphoketolase, transaldolase, transketolase, fructose 1,2-bisphosphate aldolase, fructose 1,6-bisphosphatase, ribulose-5-phosphate epimerase, ribose-5-phosphate isomerase, and trios phosphate isomerase, DHAS, and DAK, may be modified to improve metabolite production or methanol utilization. The gene may be engineered to be under the control of an inducible promoter, for example, a formaldehyde or methanol responsive promoter, a lactose inducible promoter, or a temperature or pH responsive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. The gene may also be engineered to modify the corresponding enzyme (e.g., MDH) to improve the enzyme's substrate specificity and optimal temperature in the non-naturally occurring microbe.

The method for producing a metabolite may further comprise fixing CO₂. The medium may be modified by containing higher levels of methanol which is more reduced than a sugar (e.g., glucose) such that more electrons may be generated under the conditions the non-naturally occurring microbe is grown. Other media modifications may also enable an enhanced availability of electrons in the cells. Such additives would be reducing agents or dyes (such as Methyl Viologen (MV) and other viologens). Such electrons may enable the non-naturally occurring microbe to grow on the medium while fixing CO₂. According to this method, CO₂ release may be reduced by at least about 20%, preferably by at least about 30-50%, more preferably up to about 75%.

For each non-naturally occurring microbe capable of growing in a medium comprising methanol, wherein the methanol contributes to a significant percentage (e.g., at least about 40%, 48%, 50%, 60%, 66%, 70%, 80%, 90%, 95%, 99%, or 100%) of the carbon source for the non-naturally occurring microbe, a method for preparation is provided. The preparation method comprises expressing heterologous methanol dehydrogenase (MDH) and heterologous ribulose monophosphate (RuMP) pathway enzymes in a non-methylotrophic microbe. The RuMP pathway enzymes may include 3-hexulose-6-phosphate synthase (HPS), 3-hexulose-6-phosphate isomerase (PHI). The non-naturally occurring microbe of the present invention may have any native formaldehyde detoxification system such as the frmRAB operon. The non-naturally occurring microbe may further contain a deletion of the frmRAB operon or deletion of a similar set of genes that code for enzymes that oxidize formaldehyde to CO formaldehyde typically for formaldehyde detoxification purposes. Preferably, the method comprises expressing heterologous MDH, heterologous HPS, and heterologous PHI.

The method may further comprise expressing heterologous pentose-phosphate pathway (PPP) enzymes in the non-methylotrophic microbe. The PPP enzymes may include phosphofructokinase (PFK), fructose bisphosphate aldolase (FBA), transketolase (TKT), transaldolase (TAL) fructose/sedoheptulose biphosphatase (GLPX), ribulose phosphate epimerase (RPE), and ribose-5-phosate isomerase (RPI).

The method may further comprise expressing one or more heterologous cyclic formaldehyde dissimilation enzymes in the non-methylotrophic microbe. The enzymes may include glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND). The non-naturally occurring microbe of the present invention may contain a deletion of the phosphogluconate dehydratase gene (edd).

The method may further comprise expressing heterologous CO₂ fixation pathway enzymes in the non-methylotrophic microbe. The heterologous CO₂ fixation pathway enzymes may include carbonic anhydrase (CA), formate dehydrogenase (FDH), formaldehyde dehydrogenase (FLD; the enzymes of the reductive tricarboxylic acid cycle such as ATP citrate lyase (ACL), 2-oxoglutarate: ferredoxin oxidoreductase (OGOR), isocitrate dehydrogenase (ICDH), and fumarate reductase (FR); the enzymes of the glycine cleavage system such as aminomethyltransferase (AMT), dehydrolipoyl dehydrogenase (LPDH), glycine dehydrogenase (GDH); and the enzymes of the non-oxidative glycolysis pathway including fructose phosphoketolase, xylose phosphoketolase, transaldolase, transketolase, fructose 1,2-bisphosphate aldolase, fructose 1,6-bisphosphatase, ribulose-5-phosphate epimerase, ribose-5-phosphate isomerase, and trios phosphate isomerase.

The method may further comprise expressing heterologous dihydroxyacetone synthase (DHAS, EC=2.2.1.3) in the non-methylotrophic microbe. DHAS is also known as formaldehyde transketolase or glycerone synthase. The non-methylotrophic microbe may further express heterologous dihydroxyacetone kinase (DAK, EC=2.7.1.29). DAK is also known as glycerone kinase.

The preparation method may further comprise introducing into the non-methylotrophic microbe a gene encoding any of the heterologous enzymes selected from the group consisting of the heterologous MDH, the heterologous RuMP pathway enzymes (e.g., HPS and PHI), the heterologous PPP enzymes (e.g., PFK, FBA, TKT, GLPX, TAL, RPI and RPE), the heterologous cyclic formaldehyde dissimilation pathway (PGI, ZWF, PGL, GND), the heterologous CO₂ fixation pathway enzymes (e.g., CA, FDH, FLD, reductive tricarboxylic acid cycle enzymes such as ACL, OGOR, ICDH, and FR, glycine cleavage system enzymes such as AMT, LPDH, GDH, non-oxidative glycolysis pathway enzymes such as fructose phosphoketolase, xylose phosphoketolase, transaldolase, transketolase, fructose 1,2-bisphosphate aldolase, fructose 1,6-bisphosphatase, ribulose-5-phosphate epimerase, ribose-5-phosphate isomerase, and trios phosphate isomerase), heterologous DHAS, and heterologous DAK. The gene may be expressed transiently in the non-methylotrophic microbe. The gene may be integrated into the genome of the non-methylotrophic microbe. The gene may be under the control of an inducible promoter, for example, a formaldehyde or methanol responsive promoter, a lactose inducible promoter, or temperature or pH responsive promoter. These promoters may be derived from a host cell (native) or exogenously, for example, the T7 phage promoter. These genes may also be under the control of non-DNA regulatory elements such as small RNA, antisense RNA, sensing RNA, temperature sensitive RNA or any combination thereof. The translation of these genes may be initiated with a range of ribosomal binding sites of varying strength. These genes may be borne on plasmids, fosmids, bacterial artificial chromosomes or be integrated into the host chromosome. These genes may be configured monocistronically or polycistronically. The non-naturally occurring microbe may also contain deletions of the fmrRAB operon and the edd gene.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate.

Example 1

Growth on Methanol and Biomass Labeling from ¹³C Methanol of E. coli Strain Carrying Plasmid pETM6PtacBst_MDH/Mg_RuMP Strain

We constructed a vector utilizing the pETM6 backbone, the Ptac promoter along with the MDH gene from B. stearothermophilus and the HPS-PHI fusion from M. gastri. These genes, as currently constructed, are in a monocistronic configuration. Cultures were pre-grown and induced using the following protocol. A single colony was picked from a LB plate and grown at 37° C. for 6 h in LB broth. These cultures were then pelleted and resuspended at an OD₆₀₀ of 0.5 in MOPS media containing 0.4% ribose and 0.1 mM IPTG for 12 h at 37° C. Samples were taken from these overnight grown cultures, lysed and analyzed for expression of both the MDH and RuMP fusion protein. Cultures induced overnight in MOPS ribose were also resuspended in fresh MOPS media containing 500 mM ¹³C methanol at an initial OD₆₀₀ 1.0. Samples were taken at 6 and 18 h for GC-MS analysis of ¹³C methanol incorporation into biomass and intracellular metabolites.

To confirm the newly constructed pETM6Ptac vectors expressed the desired genes, we induced the strain containing the MDH and RuMP fusion overnight in MOPS plus ribose and IPTG and analyzed the cell lysates. We were able to clearly observe the MDH (35 kDa) and the RuMP (41 kDa) (FIG. 3), indicating that both proteins were robustly expressed via the new ptac vectors. To analyze the functionality of these proteins, we transferred the induced ΔfrmA strain expression the MDH and RuMP fusion into MOPS plus 500 mM ¹³C methanol and incubated these cultures were 18 h. We were able to observe incorporation of the methanol into biomass and intracellular metabolites by the recombinant E. coli (FIG. 4). Specifically, we detected labeling in amino acids and TCA cycle intermediates (FIG. 4A). Furthermore, we were able to observe multiple carbon labeling on these metabolites (FIG. 4B), which shows that the pathway is cycling back through the pentose phosphate pathway to regenerate ribulose-5-phosphate, which is similar to native methylotrophs. This has never been demonstrated before and unequivocally supports the claims of this invention that methanol can support cell growth of the synthetic methylotroph by giving rise to core metabolites in the cells needed for cell growth.

To show that this takes place at various and lower methanol concentrations, we carried out labeling experiments using 150 mM and 250 mM methanol as the sole carbon source. We were, again, able to detect similar levels of metabolite labeling when compared with cultures incubated with 500 mM methanol (FIG. 5A). Methanol consumption coincided with the incorporation of labeled methanol into biomass, with approximately 15 and 18 mM methanol being consumed by the recombinant E. coli strain incubated in 150 and 250 mM methanol, respectively (FIG. 5B).

Example 2

Methanol is Incorporated into Biomass and Supports Cell Growth. Methanol as a Co-Substrate to Improve the Capability of Synthetic Methylotrophic E. coli to Produce More Reduced Products Such as Alcohols, Carboxylic Acids and Hydrocarbon Molecules

Because methanol is more reduced than most sugars typically used as fermentation substrates, use of methanol as a co-substrate will result in the production of more reducing equivalents compared to most sugars and will thus lead to better yields for producing metabolites like alcohols, carboxylic acids and hydrocarbons. We thus hypothesized that methanol could be metabolized by the recombinant E. coli strain when grown in the presence of additional carbon sources. After overnight induction in MOPS plus ribose, cultures were transferred to MOPS containing a mixture of methanol (500 mM) and glucose (2 mM). The strain expressing the genes for methanol utilization was able to grow to a higher OD₆₀₀ and with a faster growth rate than the empty vector control (FIG. 6A). Additionally, we were able detect labeling of intracellular metabolites under these culture conditions, indicating that the methanol is still being consumed in the presences of additional nutrient sources (FIG. 6B). Taken together, these data show that methanol is metabolized effectively in the presence of sugars as a carbon and energy source and that it enhances the growth rate and overall yields of the synthetic methylotrophic strain which is able to convert methanol to biomass and metabolites.

Example 3

Methanol Utilization Supports Cell Growth in Growth Media with Typical but Diluted LB Composition

Media of industrial fermentations typically contain supplements like yeast extracts and protein hydrolyzates. Heavy use of such supplements have been used in several celebrated new metabolically engineered strains like for the production of i-butanol and the use of reverse beta oxidation to support growth and metabolite production. Here we used a very diluted (1:4) LB broth to show that methanol is utilized without any sugars present and supports cell growth. Standard LB broth contains 10 g/l tryptone and 5 g/l yeast extract to supply carbon and energy for growth. For the growth experiments in methanol, we used MOPS media that supplemented with 2.5 g/l tryptone and 1.25 g/l yeast extract (1:4 LB:MOPS) in addition to 500 mM methanol to assay for growth and methanol incorporation.

When the MeOH utilizing strain was grown in a 1:4 mixture of LB:MOPS, we were able to observe a 68 mM decrease in methanol concentration, which contrasts to the 34 mM decrease in methanol observed in the empty vector control (FIG. 7). The observation that methanol was decreasing in the negative control was likely due to a combination of high starting OD (˜1.0) and non-specific methanol oxidation by the alcohol dehydrogenases present in E. coli (FIG. 7). These data, combined with the data from (FIG. 6B) which shows methanol incorporation in the presence of dilute LB, indicate that methanol is utilized by the cells to support cell growth without the need for added sugars.

Example 4

Methanol Growth Under Anaerobic Conditions

With the ultimate goal of using methanol to be used as a substrate for producing various metabolites such as alcohols (e.g., butanols) or mono- or dicarboxylic acids or other molecules that can be produced by metabolically engineered E. coli and other organisms typically under anaerobic or almost anaerobic conditions, we set out to examine methanol utilization under anaerobic conditions. Cultures were pre-grown and induced as outlined earlier. After overnight induction, cultures were transferred sealed anaerobic bottles containing MOPS+500 mM methanol and 2 mM glucose. We also examined the effect of methyl viologen on anaerobic growth in the presence of methanol. Methyl viologen as previously been demonstrated to consume NADH in E. coli (which should promote methanol oxidation) and induce the pentose phosphate pathway, which should promote regeneration of ribulose-5-phosphate.

When we compared growth of the methanol utilizing strain anaerobically in either glucose alone or glucose+methanol, we did not observe a difference in growth. However, supplementation with 0.15 mM methyl viologen lead to higher overall yields when cells were grown in the presence of methanol (FIG. 8). Additionally methyl viologen had no effect on growth in glucose alone. These results show that methyl viologen enhances growth in the presence of methanol and that it is a useful culture supplement to increase the titers of various metabolites that can be produced from E. coli using methanol as carbon and energy and electron source. Anaerobic growth could be reinforced through successive feedings of small quantities of glucose, and again, when the strain was grown in the presence of methanol and glucose, it grew to higher OD₆₀₀ compared when the strain was grown in glucose alone (FIG. 8). This shows that methanol can be utilized anaerobically on batch, fed-batch and continuous mode to support the production of various metabolites from recombinant E. coli and other organisms.

Example 5

Additional, Novel Metabolic Engineering E. coli to Further Enhance Methanol Utilization

We devised an additional, novel metabolic engineering strategy to enhance methanol utilization by E. coli. Aside from the main 2 main RuMP genes (PHS and PHI; FIG. 2), it is necessary for methylotrophs to be able to regenerate ribulose-5phosphate, which is the substrate used to fix formaldehyde. Ribulose-5-phoshate is an intermediate of the pentose phosphate pathway, and, in naturally occurring methylotrophs, this pathway is positively regulated by the presence of formaldehyde, which is present during growth on methanol. In E. coli, however, the pentose phosphate pathway is regulated by the ratio of NAD⁺/NADH in the cells and by the global regulator SoxS. Therefore, it is possible that the genes needed for ribulose-5-phosphate regeneration are sub-optimally regulated/expressed in E. coli during growth on methanol. Further engineering of this pathway will enhance methanol utilization and drive production of the desired end product.

There are several variants of the RuMP pathway, based upon the route of ribulose-5-phosphate regeneration. The Entner-Doudoroff (ED) variant of the RuMP pathway (FIG. 9A) possess the following stoichiometry: 3HCHO+NAD⁺→Pyruvate+NADH

Another version of the RuMP pathway is the fructose 1,6-bisphosphate (FBP) variant (FIG. 9B), which possess the following stoichiometry: 3HCHO+ADP+NAD⁺→Pyruvate+ATP+NADH

Thus, directing carbon through the FBP variant will lead to more energy for the recombinant E. coli strain, especially under anaerobic conditions, where ATP is not generated via oxidative phosphorylation. E. coli natively possesses all of the genes required for both the ED and FBP RuMP variants. We have shown above that our recombinant E. coli strain is able to regenerate ribulose-5-phosphate based on our ability to observe multi-carbon labeling of intracellular metabolites. We hypothesize however that the recombinant E. coli is likely using the less energy efficient ED pathway to regenerate ribulose-5-phosphate and generate pyruvate. While the genes for the FBP variant are present in E. coli, these enzymes favor the glycolytic direction (away from ribulose-5-phosphate) compared with the enzymes found in native methylotrophs, which favor the gluconeogenic direction (toward ribulose-5-phosphate). To this end, we have cloned the following genes associated with ribulose-5-phosphate regeneration from the B. methanolicus: fba, fructose 1,6-bisphosphate aldolase; pfk, phosphofructokinase; tkt, transketolase; rpe, ribulose-5-phosphate epimerase; and glpX, fructose 1,6-bisphosphatase. These genes have been cloned and expressed in the pCDM4 vector (FIG. 10). We used repeated growth and labeling experiments with the strain expressing the methanol utilization genes and the ribulose-5-phosphate regenerating genes to show superior methanol utilization and cell growth.

Example 6

Using Methanol to Generate Additional Reducing Equivalents

Many native methylotrophs also possess what is known as a RuMP dissimilation pattern. This pathway uses the fructose-6-phosphate generated by HPS and PHI enzymes and sequentially converts it 6-phosphogluconate, which is then converted to ribulose-5-phosphate. This process yields 2 NADPH and 1 CO₂. It is believed that methylotrophs (especially those that use the ED RuMP pathway) use this dissimilation pathway to generate the reducing equivalents used to power the electron transport chain. Again, the genes for this dissimilation pathway are present in E. coli, just likely not regulated by methanol as would be the case for native methylotrophs. To this end, we will overexpress the first gene in this pathway, glucose-6-phosphate dehydrogenase, which will force some of the fructose-6-phosphate through the dissimilation pathway. Since 6-phosphogluconate is the branching point in E. coli between the pentose phosphate pathway (desired) and the Entner Doudoroff pathway (undesired), we will also delete the first gene in the ED pathway, 6-phosphogluconate dehydrogenase. This will ensure the carbon flux is through the dissimilatory pathway. Engineering of the pentose phosphate pathway to perform the assimilatory and dissimilatory RuMP pathway is novel for E. coli and results in a recombinant strain capable of using methanol more efficiently a sole carbon and energy source.

Example 7

High-Throughput Assays for Protein Engineering MDH Proteins: Using FACS (Fluorescence-Activated Cell Sorting) for Screening MDH Libraries

We hypothesize that one potential limitation for generating a methylotrophic strain of E. coli is the ability of the NAD-dependent MDHs to efficiently oxidize methanol at the lower temperatures E. coli requires to grow. Thus we use protein engineering of the MDHs either via family shuffling or error prone PCR and generating libraries with these mutagenized copies. This requires a method to easily and in a high throughput manor screen for MDH activity in E. coli. We developed a flow cytometry based technique for screening the MDH library for desirable mutants based on formaldehyde production. This strategy utilizes a dual plasmid reporter system: one plasmid, pETM6_Ptac_MDHlib contains the MDH library, and the other plasmid, pCDM4_Pfrm_GFP contain a Green-fluorescent protein (GFP) gene under the control of a formaldehyde responsive promoter. An illustrative example of this concept is presented in FIG. 11. Upon MDH library expression with IPTG, MeOH will be added and oxidized to HCHO by active MDH mutants. The amount of GFP expression, which will correlate to HCHO production (FIGS. 12-13), will then to be utilized to select MDH mutants with higher activity via fluorescence-activated cell sorting (FACS). As seen in FIG. 14, this method can be used to detect a wide range of methanol concentrations.

Example 8

Reversibility of the Formate Dehydrogenase Activity as Tested In Vivo by ¹⁴C Incorporation Assay

As illustrated in FIG. 2, the proposed Scheme 1 for CO₂ fixation depends on the reversibility of both the formate dehydrogenase (FDH) and formaldehyde dehydrogenase (FDdH) activity along with the overexpression of a carbonic anhydrase (CA) in order to achieve high intracellular levels of CO₂. In the present work we were able to show incorporation of ¹⁴C from labeled sodium bicarbonate into the cell biomass. The possible path of the ¹⁴C incorporation is the following: NaH¹⁴CO₃+HCl→NaCl+H₂ ¹⁴CO₃ H₂ ¹⁴CO₃→H₂O+¹⁴CO₂ ¹⁴CO₂+NAD(P)H→H¹⁴COOH+H₂O+NAD(P)H (reverse formate dehydrogenase activity)

In more detail, the labeling experiment was performed as follows: Single overexpression E. coli colonies of Candida boindini FDH and Clostridium carboxidivorans FDH (selenocysteine codon was substituted with cysteine codon) along with the empty vector control (pACYCDuet-1) were grown overnight at 37° C. in TB broth. The cultures were used the next day to inoculate larger TB broth cultures. When the cultures reached an OD₆₀₀ of 0.5, 0.1 mM IPTG and 5 ul of 1 mCi/ml ¹⁴C sodium bicarbonate was added and the cultures were transferred to serum bottles in order to grow anaerobically for 4 h. After 4 h the culture was treated with H₂SO₄ to release any unincorporated ¹⁴C with the form of ¹⁴CO₂. Whole cells were then harvested by centrifugation and the cell pellets were treated with 100 mM Na—K phosphate buffer (pH3.0). After centrifugation the cell pellets were resuspended in a solution of 0.1M sodium dodecyl sulfate and 0.2N NaOH. 1 ml of the resuspended solution was heated at 90° C. for 2 h. Incorporation of ¹⁴C into the cell biomass was analyzed by liquid scintillation.

We were able to detect labeling in the cell mass that was higher for both the Candida boindini and the Clostridium carboxidivorans FDH compared to the empty vector control (FIG. 15). Background levels of CO₂ incorporation were expected because of CO₂-pyruvate exchange under the anaerobic conditions.

The above described in vivo labeling experiment showed ¹⁴C incorporation into the cell biomass suggesting possible reverse formate dehydrogenase activity under anaerobic conditions.

Example 9

Overexpression of Selenocysteine-Containing Formate Dehydrogenases

In most cases, the formate dehydrogenases that act as part of the Wood-Ljungdahl pathway for CO₂ fixation in acetogens, are characterized by the presence of selenocysteine in their active site. It has been shown that substitution of the selenocysteine by cysteine in these enzymes reduce their enzymatic activity by 90%. The incorporation of selenocysteine in selenoproteins requires the recruitment of specialized enzymes and t-RNA. In addition the corresponding RNA contains a characteristic structural element, namely SECIS element. The proteins related to this mechanism are encoded by a set of genes, namely selA-D. The codon responsible for the selenocysteine incorporation is UGA, identical with the universal stop codon. This codon when followed by a specific hairpin structure of the RNA, the so-called SECIS element, is not recognized as a STOP codon but instead recruits the necessary machinery for selenocysteine incorporation. The mechanism appears to be highly specific for each organism with SECIS elements of different structure responsible for the recruiting of the appropriate enzymatic machinery in different species.

We explored the heterologous expression of the Moorella thermoacetica (Moth) selenocysteine containing FDH-A subunit together with the FDH-B subunit in E. coli. The two subunits together when purified were previously shown to have CO₂ reductase activity in vitro. To test this activity in vivo we designed a hybrid cDNA sequence that was both codon-optimized for E. coli expression, as well as had a substituted Clostridia SECIS element with a potential E. coli recognizable one.

The recombinant hybrid Moorella FDH-A, as designed by two different companies (Genscript and DNA2.0) with the E. coli recognizable SECIS element, was expressed in E. coli together with FDH-B. While the FDH-B overexpression band had the expected size of approximately 75 kD, FDH-A was expressed only as a truncated protein of 25 kD (FIG. 16) instead of 100 kD that would be the size of the full-length selenoprotein. This finding suggests that the E. coli SECIS element itself cannot achieve selenocysteine incorporation.

Considering the above findings, we pursue two alternatives strategies to achieve selenocysteine incorporation into formate dehydrogenases heterologously expressed in E. coli. First, we pursue additional mutations at the immediately adjacent sequence of the SECIS element which could help with selenocysteine incorporation. Alternatively, we express the Moorella FDH-A with the native Moorella SECIS element in parallel with expression of the native Moore/la selenocysteine-related genes and more specifically selB and selC coding for the selenocysteine-specific elongation factor and the t-RNA(sec).

Example 10

In Vivo MeOH Oxidation and HCHO Reduction Activity of MDHs

Nine NAD⁺-dependent MDHs (all codon optimized for E. coli) from Bacillus spp. have been cloned and expressed in E. coli. The nine synthesized MDHs are listed in Table 1.

In addition to in vitro activity disclosed earlier, all nine MDHs exhibit in vivo methanol oxidation activity in E. coli BL21(DE3) strains (FIG. 17) but only eight possess in vivo activity in the E. coli ΔfrmA strain, which lacks the native FrmRAB system for formaldehyde oxidation (FIG. 18). In E. coli the enzymes that perform the formaldehyde degradation are encoded by a three gene operon namely, fmrRAB. frmR encodes a transcriptional repressor of the operon, frmA encodes the S-hydroxymethylglutathione dehydrogenase and frmB encodes the S-formylglutathione hydrolase. Note that E. coli BL21(DE3) strains contain active formaldehyde dehydrogenase that introduces competition for formaldehyde, suggesting the intracellular formaldehyde is detoxified to CO₂ during the course of the experiment. To eliminate formaldehyde competition, all single gene and co-expression constructs were transformed into an E. coli ΔfrmA strain using a modified pETM6 vector containing the tac promoter (see Examples 15 and 17 below). While in vivo activity in E. coli has been reported previously for five of these MDHs (Bme MGA3 Mdh, Bme MGA3 Mdh2, Bme MGA3 Mdh3, Bme PB1 Mdh, and Bme PB1 Mdh2), the data for in vivo activity in E. coli for Bst 2334 Mdh, Bst NUB3621 Mdh, Bme C1 Mdh, and Bme PB1 Mdh1 are new. As presented, Bme MGA3 Mdh2 and Bme MGA3 Mdh3 possess the highest activity in vivo in E. coli BL21(DE3) strains while Bst 2334 Mdh and Bme MGA3 Mdh3 possess the highest activity in vivo in E. coli ΔfrmA strains. Furthermore, MDH activator proteins do not exhibit a stimulatory effect in vivo when co-expressed with their respective MDHs in E. coli BL21(DE3) strains, suggesting the physiological role of these activator proteins within methylotrophic growth remains unknown.

In vivo HCHO reductase activity of all MDHs has been examined in addition to methanol oxidation. These data show that the rate of HCHO reduction is greater than that of methanol oxidation (FIG. 19). These results are explained by examining the thermodynamics of the system, which suggests that methanol oxidation is unfavorable under standard conditions (Δ_(r)G=+34.2 kJ/mol) but becomes slightly favorable under physiological conditions (Δ_(r)G=−1.0 kJ/mol) as the intracellular HCHO concentration remains at micromolar levels.

Example 11

Optimized Methanol Utilization in One or Sequential Bioreactors with Optimized Temperature for Optimal MDH Activity

Thermodynamic calculations show a positive change in reduction potential and negative change in reaction Gibbs free energy when calculated using estimated physiological conditions (Table 2). This suggests that MeOH oxidation via NAD-dependent MDH enzymes becomes more favorable at higher temperatures, i.e., the optimal growth temperature of 45-55° C. of these thermophilic bacillus methylotrophs. Specifically, the change in the reaction Gibbs free energy (Δ_(r)G) decreases from −1.0 to −2.9 kJ/mol as the temperature increases from 37 to 55° C., suggesting a more favorable reaction at higher temperatures. This further demonstrates that MeOH oxidation via NAD-dependent MDH enzymes is plausible under the physiological growth conditions of mesophiles such as E. coli. As MeOH oxidation via NAD-dependent MDH enzymes is favorable only to a small extent under physiological conditions compared with the other methanol oxidation systems, the immediate consumption of HCHO via an assimilation pathway will increase the favorability of MeOH oxidation. For example, at 37° C., when the concentration of HCHO is lowered from 0.17 to 0.017 mM, the change in the reaction Gibbs free energy decreases from −1.0 to −7.0 kJ/mol while the equilibrium constant shifts from 0.925 to 8.5, thus becoming much more favorable. Therefore, increased methanol oxidation within synthetic methylotrophic organisms may be achieved by keeping the intracellular HCHO concentration at very low levels, likely through its immediate consumption via a HCHO assimilation pathway.

For the aforementioned and cloned MDHs, the rate of methanol oxidation increases with increasing temperature. As demonstrated in FIG. 20, the in vivo methanol oxidation rate doubles as the growth temperature is increased from 37 to 40° C. These data support the thermodynamic calculations shown in Table 2 and demonstrate that the rate of methanol oxidation would be increased in an E. coli strain adapted for growth at higher temperatures. This is a realistic possibility as has been reported. Thus, we developed cultivation strategies to take advantage of these optimal conditions for MDH activity. E. coli strains (WT or mutant strains such as E. coli ΔfrmA strains) are developed by evolutionary adaptation to grow at any higher temperature such as 40° C., 45° C. or 50° C. These strains are then used to engineer the methanol utilization pathway described here in combination with any engineered pathway to produce a desirable oxychemical as shown conceptually in FIGS. 1 and 2. The expressed genes are optimized for enzyme expression and activity at the higher temperature. These engineered strains are used either in a single bioreactor operating at a higher temperature (e.g., 40° C., 45° C. or 50° C.), or in reactors in series, each operating in a different temperature so that one can separately optimize cell growth and product formation. For example, cells are grown in one reactor at 37° C. and then product is formed in a second bioreactor operating at 40° C., 45° C. or 50° C. One could use three or more bioreactors sequentially or otherwise, each operating at a different temperature.

Example 12

Protein Engineering to Generate Desirable MDH Mutants

The chosen MDHs may exhibit a limitation in the context of a desirable product. From our data shown in FIG. 21, all MDHs possess higher activity towards butanol compared with methanol in vitro. So, if the desirable product is butanol this might be a problem. This can be resolved as disclosed here. Our data (FIG. 21) show that the different MDHs exhibit a wide range of alcohol selectivity, suggesting this natural variability could be used in a directed evolutionary protein engineering approach. To eliminate activity and selectivity towards butanol, a protein engineering approach involving successive rounds of DNA shuffling and/or error-prone PCR is performed. Using such protein engineering approaches, for different enzymes (but NOT for MDHs) it has been possible to alter substrate specificity and increase activity towards specific substrates. To accomplish this engineering strategy, an accurate high-throughput screening technique is required. Data for aldehyde indicator plates has yielded positive results to date as illustrated in FIG. 22. These plates contain a mixture of pararosaniline and sodium bisulfite that reacts with aldehydes to form a red color. To date, these plates have only been examined with E. coli BL21(DE3) MDH strains. Better results are to be obtained with E. coli ΔfrmA strains since the formaldehyde generated from methanol oxidation will not have the potential to escape to CO₂. As background color presents an issue for these plates, a flow cytometry-based selection approach will be used in place of or in addition to aldehyde indicator plates. Operating of the same principle, i.e., the reaction of aldehydes with pararosaniline and sodium bisulfite, a flow cytometry-based method will select clones having desirable properties. A selection strategy may also be implemented for identifying desirable MDH mutants. For selection, the MDH library would be cloned with formaldehyde assimilation genes, e.g., HPS and PHI, and introduced to a selective pressure such as a minimal media containing only methanol and butanol. Ideally, a mutant with increased methanol oxidation activity that has lost activity towards butanol would be selected for. Furthermore, these MDHs can be used in synthetic methylotrophic strains that are aimed at producing chemical other than higher alcohols, such as fatty acids, biodiesel, organic acids, carboxylic acids, or butanediol.

Example 13

Generation of a Methanol Consuming Strain of E. coli with Glucose Present as a Co-Substrate

We previously generated a methylotrophic strain of E. coli via expression of a non-native methanol dehydrogenase from B. stearothermophilus in conjunction with the two genes of the ribulose monophosphate (RuMP) pathway: 3-hexulose-6-phosphate synthase (HPS, 2 genes) and 6-phospho-3-hexuloisomerase (PHI) from the methylotroph strain L3 using the pETM6 vector. This strain could grow in media containing methanol and ribose with observed methanol consumption. We have redesigned the vector so that the genes are now in a monocistronic operon configuration as we did not observe robust expression of the MDH protein while in the pseudo-operon configuration. We repeated the growth analysis with this new strain and observed a decrease in MeOH concentration at a rate of approximately 3.5 mM h⁻¹, whereas the MeOH concentration in the empty vector control remained fairly unchanged (0.5 mM h⁻¹) (FIG. 23A). Both strains exhibited an increase in optical density (OD) at 600 nm in media containing methanol and glucose with the strain expressing MDH and the RuMP pathway reaching a slightly higher OD (FIG. 23B). Because the empty vector control strain was able to grow under these conditions, and because the increase in OD of the strain expressing the recombinant MeOH utilization pathway is just a little greater than the control, we hypothesized that the majority of the MeOH was not going into biomass but was rather dissimilated to CO₂ via an endogenous formaldehyde detoxification pathway.

Example 14

Deletion of the Native System for Formaldehyde Oxidation to Enable Better Strain Engineering

In order to achieve a strain capable of consuming methanol as a carbon- and energy-source, we ordered a frmA deletion strain of E. coli from the Keio collection, which disabled the native E. coli formaldehyde detoxification system (FrmRAB). We grew the ΔfrmA strain, the isogenic wild type BW25113, and a ΔfrmA strain expressing the M. gastri HPS-PHI fusion gene on pUC19 (ΔfrmA/pUC_Mgfusion) in medium containing formaldehyde, and followed the disappearance of formaldehyde. We found that the wild-type strain exhibited robust detoxification of formaldehyde at a rate of approximately 0.47 mM h⁻¹. In contrast, the ΔfrmA strain was essentially unable to detoxify formaldehyde (0.07 mM h⁻¹). The ΔfrmA/pUC_Mgfusion strain was able to consume formaldehyde at a rate of 0.3 mM h⁻¹ (FIG. 24). This data confirm the hypothesis that the native E. coli FrmAB genes are very efficient at dissimilating formaldehyde and are likely able to outcompete the recombinant RuMP genes. Thus, we use the ΔfrmA strain as the host strain for the recombinant MeOH pathway for all future experiments and strain developments.

Example 15

Expression Based on the Inducible Tac Promoter to Facilitate Regulated Gene Expression for Optimal Pathway Engineering

The pETM6 vector is part of the Biobricks family of vectors that relies on isocaudomer pairs to sequentially add genes in a pathway to the vector. In order to continue to make use of this vector for cloning and expressing multiple genes on a single plasmid, we must now account for the fact that the ΔfrmA does not carry the gene for the T7 polymerase and is thus incompatible with the T7 promoter. Therefore, we have redesigned these vectors to contain the tac promoter instead, which would allow for robust IPTG inducible gene expression in the ΔfrmA host strain (FIG. 25).

Example 16

Engineering a Formaldehyde-Inducible Promoter System in E. coli

The RuMP pathway relies on the pentose phosphate pathway for the regeneration of the ribulose-5-phosphate needed to act as the acceptor for formaldehyde fixation. In E. coli, the pentose phosphate pathway is primarily regulated by the availability of reducing equivalents (NAD+/NAHD) whereas in methylotrophic organisms, the pentose phosphate pathway is regulated by the presence of formaldehyde. Therefore, we sought to identify and use a promoter that can respond to formaldehyde to the genes of the E. coli pentose phosphate pathway which will ensure the cells maintain an appropriate ribulose-5-phosphate pool. It has previously been determined that the frmRAB operon responds to increasing formaldehyde concentration indicating that E. coli should contain a native formaldehyde inducible promoter and all the machinery required to transcribe it.

To examine this, we removed the T7 promoter from the pETM6 vector and replaced it with the 200 bp region directly upstream of the frmRAB operon. We then cloned GFP into the multiple cloning site of the vector and transformed this new vector (pETM6_pFrm_GFP) into a generic cloning strain of E. coli. This strain was induced with formaldehyde or left uninduced and analyzed for GFP expression. GFP could be detected in the induced and uninduced cultures of pETM6_pFrm_GFP at the initial time point. The amount of detectable GFP increased for the cultures that were induced by formaldehyde and remained constant for the uninduced strains (FIG. 26). There are two possibilities for being able to detect GFP in the uninduced cultures: 1) the frmRAB promoter could be leaky, thus leading to GFP expression, or 2) there is a small amount of endogenous formaldehyde present in the cytosol of bacteria which leads to a small amount of expression from the promoter. However, we were able to detect robust expression of GFP after induction with formaldehyde. Therefore, this promoter responds to formaldehyde and can be used to upregulate the pentose phosphate pathway and other genes that might be necessary for growth using C1 carbon sources, since formaldehyde will always be present in the strain oxidizing methanol to formaldehyde.

Example 17

Metabolic Engineering to Enable E. coli to Grow Effectively on MeOH as Sole Carbon Source

E. coli BW25113 and JW0347-1 were grown using methanol as the sole carbon source. Codon-optimized methanol dehydrogenase genes (coding for MDH 2 and MDH 3) from Bacillus methanolicus MGA3 were cloned with and without the corresponding activator gene into a modified pETM6 ePathBiobrick vector with the T7 promoter replaced by the tac promoter (pETM6-tac), enabling use in any E. coli host strain (FIG. 27). Codon optimized hexulose phosphate synthase gene (coding for HPS) and codon optimized hexulose phosphate isomerase gene (coding for PHI) from M. gastri were cloned into a modified pACM4 ePathBiobrick vector with the T7 promoter replaced by the tac promoter (pACM4-tac). The codon optimized HPS and PHI were cloned in a monocistronic orientation (FIG. 27). Two fusion HPS and PHI genes, from M. gastri and strain L3, were cloned in pACM4-tac as well (FIG. 27). pETM6-tac and pACM4-tac have compatible origins of replication and different antibiotic resistance markers, enabling co-transformation. These constructs containing an MDH gene and the HPS and PHI genes, when co-transformed, enabled sustained growth on methanol in minimal medium.

Example 18

Employing a Mutation-Directed Evolution Selection Strategy on Methylotrophic E. coli Yields Fast Growing Methanol Consumer

E. coli BW25113 and JW0347-1 containing plasmids with genes encoding the HPS and PHI genes or a combination of MDH, HPS, and PHI genes were mutated using the chemical NTG (n-methyl-n′-nitro-n-nitrosoguanidine). These populations of mutants were allowed to recover and then transferred to a selection of minimal medium with formaldehyde or methanol as the sole carbon source. Growth was observed over multiple generations and serial transfers. Individual mutants were isolated and screened for improved growth. Multiple mutants were found to have superior growth rates and higher cell densities when grown on methanol or formaldehyde compared to their parent strain.

Example 19

Demonstrating Functionality of the Engineered RuMP-Based Methylotrophic Pathway by Showing Incorporation of Carbon from Formaldehyde into Biomass Components and Alter CO₂ Production Using ¹³C Labeling and Flux Analysis

Using ¹³C-labeled substrates, we can determine how effective these new pathways are by examining the extent of incorporation of these substrates. Experiments were performed with four E. coli strains:

i. an unmodified E. coli strain containing an empty vector (pM6; control)

ii. an unmodified E. coli strain containing a pUC19 vector expressing the M. gastri RuMP fusion genes.

iii. The ΔfrmA E. coli strain where as described above the native formaldehyde oxidation system has been inactivated.

iv. The ΔfrmA E. coli strain containing a pUC19 vector expressing the M. gastri RuMP fusion genes.

Cultures were inoculated at an OD₆₀₀ of 0.500 in minimal MOPS media with 0.5 mM ¹²C-formaldehyde, as an unlabeled control, or ¹³C-Formaldehyde. After 2 hours, the same amount of formaldehyde was added again to each sample. After 4 hours, the headspace gas was analyzed by mass spectrometry. The formaldehyde concentration in the media was quantified and biomass samples were analyzed for labeling in intracellular metabolites by GC-MS.

FIG. 28 shows the amount of labeled carbon dioxide in the headspace, while FIG. 29 shows the formaldehyde left in the media. The amount of formaldehyde utilized and CO₂ produced from formaldehyde oxidation was high in the unmodified E. coli strains. In contrast, in the ΔfrmA E. coli strain, very little formaldehyde was utilized and very little CO₂ was produced, as would be expected. In the ΔfrmA E. coli strain expressing the M. gastri RuMP fusion genes, a good amount of formaldehyde was utilized, yet very little CO₂ was produced, thus suggesting that formaldehyde utilization did not result in oxidation to CO₂ but rather its incorporation into biomass. To support further this conclusion, FIGS. 30 and 31 show the extent of labeling from ¹³C-formaldehyde into key metabolites of the central carbon metabolism. Compared to the unlabeled controls, the data show that ¹³C-formaldehyde is being highly incorporated into these metabolites, and thus very little formaldehyde is oxidized into CO₂. This shows that the engineered RuMP pathway is functional and capable of incorporating carbon from C1 compounds at the oxidation state of formaldehyde.

Example 20

Metabolic Engineering to Make E. coli Fix CO₂ when Growing on MeOH

In order to engineer E. coli to fix CO₂ when growing on MeOH, we explore the scheme where CO₂ is reduced to formate and subsequently to formaldehyde through the reverse activity of a formate dehydrogenase (FDH) and a formaldehyde dehydrogenase (FLD), respectively. The co-expression of a carbonic anhydrase (CA) is also employed to increase the availability of CO₂ within the bacterial cells.

A. Formate Dehydrogenase Expression

In our invention the CO₂ fixation route starts with the conversion of CO2 to formate through a reversed formate dehydrogenase activity. In this context we explore heterologous expression of the Moore/a thermoacetica (Moth) FDH-A subunit and FDH-B subunit in E. coli. We have designed a hybrid cDNA sequence that is both codon-optimized for E. coli expression, as well as has a substituted Clostridia SECIS element with a potential E. coli recognizable one.

The final optimized sequence for higher expression levels of the Moth FDH-A in E. coli with the artificially designed SECIS element designed by DNA2.0 is shown in FIG. 32A (SEQ ID NO: 1).

The SECIS substitution resulted in a modified ORF with two amino-acid changes (highlighted in bold and underlined letters) in the encoded protein (Table 3).

For the previously mentioned CO2 fixation scheme, we also test a different formate dehydrogenase that does not have a selenocysteine as part of its active site. For that we designed a codon-optimized Candida boindini formate dehydrogenase for over-expression in E. coli. The optimized sequence designed by Genscript is shown in FIG. 32B (SEQ ID NO: 2).

The C. boidini formate dehydrogenase is cloned into pACYC-Duet-1 vector that allows overexpression under the IPTG inducible T7 promoter.

Moth DNA2.0 designed FDH-A is cloned into pCOLA-Duet-1 vector together with FDHB designed from Genscript, in order to achieve co-expression of the two subunits (FDH-A, FDH-B) proteins that constitute the active form of the Moth formate dehydrogenase protein complex.

The reversibility of the enzymatic activity in both cases is checked in vivo according to the assay:

(a) Start culture and incubate at 37° C. overnight in LB media. Inoculate fresh TB media and incubate until OD₆₀₀=1-2. Spin down the bacteria at 4000 g for 10 min at 4° C.;

(b) Resuspend bacterial pellet in minimal media (M9, MOPS) supplemented with glucose or glycerol;

(c) Induce with 0.2-1 mM IPTG and transfer the culture into serum bottles to be cultivated anaerobically;

(d) After 1-2 hours of incubation at 37° C. add 1 mM methyl-viologen to maintain the media reduced;

(e) Addition of sodium bicarbonate to provide the substrate for the in vivo activity; and

(f) Let the culture grow at 37° C., sample at 1-18 h and test for protein expression, formate and formaldehyde concentration.

B. E. coli Strain BL21 (DE3) by Knocking Out the Native Operon Responsible for Formaldehyde Detoxification (frmRAB)

In bacteria the detoxification of the highly toxic formaldehyde is essential for survival. The formaldehyde detoxification pathway has been characterized in E. coli and is found to involve a glutathione (GSH)-dependent NAD-linked formaldehyde dehydrogenase (GSH-FDH) and a formyl-GSH hydrolase (FGH). In more detail, formaldehyde spontaneously reacts with GSH to produce S-hydroxymrthylglutathione, which is then oxidized to S-formylglutathione by formaldehyde dehydrogenase. In E. coli the enzymes that perform the formaldehyde degradation are encoded by a three gene operon namely, fmrRAB. frmR encodes a transcriptional repressor of the operon, frmA encodes the S-hydroxymethylglutathione dehydrogenase and frmB encodes the S-formylglutathione hydrolase.

In order to test the reversibility of formate dehydrogenase and formaldehyde dehydrogenase in vivo, the formaldehyde detoxification pathway has to be eliminated because it dominates the fate of any formaldehyde produced into the engineered bacterial system. For that reason, we have designed an experiment to knock-out the frmRAB operon in E. coli BL21(DE), the strain we routinely use for T7 promoter driven, IPTG-inducible protein overexpression.

The sequence of frmRAB operon in E. coli BL21(DE3) is shown in FIG. 32C (SEQ ID NO: 3).

The PCR-based process used includes a one-step inactivation of the three chromosomal genes as described by Datsenko et al, 2000 (PNAS 97(12): 6640-6645), The primers used that have 40-nt extensions are homologous to the regions adjacent to the frmAB operon (underlined) are the following:

H1P1: (SEQ ID NO: 6) ATATAGCATACCCCCCTATAGTATATTGCGTGCAGATAATGAGGTGCGAA ATTCCGGGGATCCGTCGACC  H2P2: (SEQ ID NO: 7) TGTAGGCCGGATAAGGCGTTCACGCCGCATCCGGCAGTCGTGCACTATTA TGTAGGCTGGAGCTGCTTCG 

The rest of the primer sequence will anneal to the pKD46 plasmid to amplify a kanamycin resistant cassette. The resulted PCR amplified fragment is then introduced into the BL21 strain already transformed with the lamda-phage Red recombinase carrying plasmid (pKD13). This allows a recombination event where the frmRAB operon is substituted with the kamamycin cassette and thus eliminated from the strain's chromosomal DNA. Introduction of the PCR20 vector into the kanamycin resistant strain eliminates the kanamycin cassette through a final recombination event. The engineered BL21 strain is expected to have significantly reduced levels of formaldehyde detoxification which will allow more precise in vivo activity measurements for the formate dehydrogenase and formaldehyde dehydrogenase overexpressing strains.

Example 21

Expression of MDHs in E. coli and their In Vivo Activity

Nine NAD⁺-dependent MDH candidates are listed in Table 4. All of the enzymes were codon optimized for E. coli except for Bacillus stearothermophilus (Bst) 2334 and cloned into the pETM6 expression vector, which were subsequently transformed into E. coli Rosetta expression strains. Of the nine enzymes, only Bst 2334 Mdh, Bst NUB3621 Mdh, Bacillus methanolicus (Bme) MGA3 Mdh2, and Bme MGA3 Mdh3 possess detectable activity in vivo compared to the pETM6 empty vector control strain as presented in FIG. 33. The in vivo assay was performed as follows: cultures were first grown in LB media at 37° C. to an optical density of 0.4, where protein expression was induced with 0.5 mM IPTG and allowed to proceed for 3 hours at 37° C. After expression was complete, cells were harvested and normalized in M9 media to an optical density of 2. The reaction was initiated with 1M methanol, and formaldehyde accumulation in the supernatant was monitored over a one hour span using the method adopted by Nash (Biochemical Journal 55(3): 416-421). Based on specific activities calculated from initial reaction rates, which is presented in FIG. 34, it appears that the Bme MGA3 MDH enzymes are best. Although co-expression of the respective ACT proteins with each MDH enzyme was achieved (see FIG. 35), the stimulatory effect of ACT was not observed in vivo as specific activities of co-expression cultures were less than those of corresponding single MDH expression strains.

To sum, a total of nine NAD⁺-dependent MDH enzymes were identified from thermophilic Bacillus spp.; of these, only four possess detectable activity in vivo in recombinant E. coli strains. These four MDH candidates are Bst 2334 Mdh, Bst NUB3621 Mdh, Bme MGA3 Mdh2, and Bme MGA3 Mdh3. Although ACT stimulates MDH activity in vitro, co-expression of ACT with the MDH enzymes decreased specific activity in vivo rather than stimulating it.

Example 22

Protein Engineering of MDHs to Enhance Substrate Specificity and Optimal Activity Temperature

As discussed, NAD⁺-dependent MDH enzymes possess higher activity and specificity for butanol than methanol. Through protein engineering, this limitation may be alleviated. Along with increasing activity and specificity for methanol, protein engineering can be also used for increasing MDH activity at 37° C. since the thermophilic Bacillus spp. from which they are derived have optimal growth temperature above 45° C.

Example 23

Generation of a Methanol Consuming Strain of E. coli

We first generated a methylotrophic strain of E. coli via expression of a non-native methanol dehydrogenase in conjunction with the two genes of the ribulose monophosphate (RuMP) pathway: 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI). To achieve this, we combined the B. stearothermophilus MDH with the HPS (2 genes) and PHI from the methylotroph strain L3 (Chu and Papoutsakis, Biotechnol. Bioeng. 29(1): 55-64) using the pETM6 vector. The pETM6 vector is part of the Biobricks family of vectors that relies on isocaudomer pairs to sequentially add genes in a pathway to the vector. Briefly, the B. stearothermophilus gene along with the 3 genes comprising the L3 RuMP pathway were amplified by PCR and cloned separately into the pETM6 vector at NdeI and XhoI sites. Single gene-containing vectors were then digested with SalI and AvrII for donor vectors or SalI and SpeI for receiving vectors. The SalI and AvrII fragment contained the T7 promoter along with the gene of interest and was subsequently ligated to the pETM6 linearized by SalI and SpeI. This ultimately culminates in a vector containing all 4 genes, each with their own T7 promoter and a single terminator of the set of genes (pseudo-operon conformation).

Example 24

Growth Analysis Utilizing Methanol as a Carbon and Energy Source

We next wanted to see if E. coli expressing MDH and genes of the RuMP pathway would be able to utilize methanol as a carbon and energy source. E. coli BL21(DE) containing either pETM6_Bs_MDH_L3RuMP or pETM6_Empty was grown overnight in LB broth plus ampicillin. These cultures were diluted 1/50 into fresh LB and incubated until the culture reached an OD₆₀₀ of approximately 0.4. At such time, the cultures were induced with 0.5 mM IPTG and incubated for a further 3 h. Cultures were grown in 10 ml of media in 50 ml conical tubes throughout the experiment. After IPTG induction, the cultures were pelleted and washed twice with M9 minimal media before being re-suspended at an OD₆₀₀ of 0.2 in M9 media containing ampicillin, IPTG, and 0.75% methanol. However, under these conditions no growth was observed by the strain carrying the genes for methanol utilization. We hypothesized that this was due to the strain's inability to regenerate ribulose-5-phosphate from fructose-6-phosphate (FIG. 1). Even though E. coli should possess the genes to natively undergo with pathway (via the pentose phosphate pathway; PPP), it is likely the native genes are not expressed under these conditions at sufficient levels. Therefore, we repeated the aforementioned experiments. This time we supplemented the M9 media with 0.04% ribose and 0.005% fucose (which has been shown previously to enhance ribose catabolism) (Autieri, Lins et al. 2007) in addition to 0.75% methanol. This time we were able to observe an increase in cell density or cell concentration (OD₆₀₀) in the strain carrying the methanol utilization genes compared with no increase in the control strains containing the empty vector (FIG. 36). After 24 h, the cultures were pelleted and re-suspended in fresh media and allowed to continue to grow. After a further 6 h, the OD₆₀₀ nearly doubled for the strain possessing the methanol utilization genes and HPLC analysis revealed that the strain had consumed approximately 5 mM of methanol (FIG. 37A). The OD₆₀₀ remained constant for the control and no methanol consumption was observed (FIG. 37B). After an additional 18 h, the OD₅₀₃ remained constant for both strains and no additional methanol was consumed during this time period. We hypothesized that this to be due to the fact that all of the ribose had been consumed for the strain carrying the methanol utilization genes during the first 6 h of growth. To test this, we added an additional 0.04% ribose and 0.005% fucose to the media to see if a spike with ribose would lead to additional methanol consumption. Indeed, for the strain carrying the methanol genes, the OD₆₀₀ increased from approximately 0.5 to 0.8 12 h after the culture was spiked with ribose (FIG. 36). Similarly, the methanol concentration decreased again, by about 5 mM for these cells as well (FIG. 37A). At this point, the cultures were again pelleted and re-suspended in fresh media with methanol, this time also containing 0.008% ribose and 0.005% fucose, to see if increasing the ribose concentration would affect methanol consumption. Six h after being re-suspended in the 2× ribose, the OD₆₀₀ of the strain containing the methanol utilization genes increased from approximately 0.5 to 1 (FIG. 36) and the amount of methanol consumed was observed to be 10 mM (FIG. 37A), indicating that doubling the ribose concentration lead to double the amount of methanol consumed by the cells. These data demonstrated that our engineered E. coli strain expressing MDH and two enzymes of the RuMP pathway (FIG. 1) can grow in minimal media containing methanol. Observed OD₆₀₀ increases are concordant with methanol depletion from the media.

Example 25

Cloning the Complete RuMP Pathway in E. coli

Given the previous data, it appears that in order for E. coli to be able to utilize methanol without stimulation with ribose and fucose, it must be able to regenerate ribulose-5-phosphate in order to utilize the RuMP pathway. To achieve this goal, we were inspired by the methylotrophic organism Bacillus methanolicus, which utilizes the RuMP pathway for growth on MeOH. In this organism, growth on MeOH is plasmid dependent, as these organisms harbor a plasmid that carries an mdh gene as well as five homologues of the pentose phosphate pathway genes (pfk, phosphofructokinase; fba, fructose bisphosphate aldolase; tkt, transketolase; glpX, fructose/sedoheptulose biphosphatase; rpe, ribulose phosphate epimerase). When the strain is cured of the plasmid, it loses the ability to grow on methanol. This has been shown to be due to the loss of the five PPP homologues vs loss of the MDH. Thus, we hypothesize that expression of the five genes from B. methanolicus in E. coli along with expression of MDH, HPS, and PHI will allow for the cells to regenerate ribulose-5-phosphate and thus grow utilizing methanol as the sole carbon and energy source. To that end, we have had the five genes from synthesized and optimized for expression in E. coli. We used the vector pCDM4, which is a medium copy vector in the Biobrick family compatible with the pETM6 vector to clone all five genes using the pseudo-operon conformation (FIG. 38). We carried out growth analysis of a BL21(DE) strain containing both vectors in MOPS minimal media containing only methanol as the carbon and energy source.

Example 26

Metabolic Engineering to Make E. coli Fix CO₂ when Growing on MeOH

To achieve this, we explore two schemes and possibly a 3^(rd) one as a backup (these are shown on FIG. 2): Scheme 1 uses the reduction of CO₂ to formate and then to formaldehyde. Scheme 2 is a modified reverse TCA (tri-carboxylic acid) pathway and Scheme 3 is based on glycine synthase. First, for all schemes, we overexpress a carbonic anhydrase (CA) in order to increase the availability of CO₂ to the cells and drive the overall reaction to HCHO formation. CA catalyzes the hydration of CO₂ to HCO₃ ⁻ and plays an important role in increasing the effective CO₂ concentration for CO₂ fixation in cyanobacteria. CA from a cyanobacterium was shown to enhance CO₂ utilization in E. coli, and thus we propose to use the same gene in our strains. We note that the physiological role of this CA is to enhance CO₂ uptake, unlike the role of the native E. coli CA, which is part of an operon induced under cyanate stress and thus has likely an opposite role. Nevertheless, the native CA did not interfere with the beneficial effect of the recombinant CA. For Scheme 1, we co-express CA with formate DH (FDH) and HCHO DH (FdDH) as a first step toward enhancing the overall CO₂ fixation. A similar strategy coupling the use of CA with a 3-enzyme pathway improved the formation of MeOH from CO₂ by >4-fold.

CO₂ fixation starts with CO₂ conversion to formate through a reversed formaldehyde dehydrogenase activity.

We heterologously express in E. coli a formate dehydrogenase from Moorella thermoaceticum that has been shown to act in the reverse direction as a CO₂ reductase. Moorella thermoacetica has been extensively studied as a model acetogene that uses the Wood-Ljungdahl pathway for CO and CO₂ fixation. In 1982, Yamamoto et al. purified and characterized the Moorella formate dehydrogenase as a heterotetramer (α2β2) consisting of two each of two different subunits, namely α and β. The calculated molecular weights for these were 96,000 and 76,000, respectively. The enzyme was also found to contain selenium (in subunit a), tungsten, iron and sulfur and to be inactivated even in trace amounts of oxygen. It catalyzed the conversion of CO₂ to formate with NADPH as cofactor. The two genes encoding for Moorella formate dehydrogenase subunits α and β, FDH-A and FDH-B were identified in 2008 upon release of the complete genome sequence of the organism with accession numbers AAB18330 and AAB18329, respectively.

The FDH-A gene was found to encode for a selenoprotein (MW=98,000) with the selenocysteine incorporation at position 358. In bacteria the SECIS element is an RNA element around 60 nucleotides long that appears soon after the selenocysteine codon (UGA) and forms a characteristic stem-loop structure. In the lack of the SECIS element the UGA sequence is recognized as a stop codon.

The SECIS element in Clostridia has a different primary and secondary structure compared to the E. coli one. Our effort focused in creating a hybrid cDNA sequence exchanging the Clostridia SECIS element with the E. coli SECIS element. Since the SECTS element is part of the ORF we tried to minimize the differences in the encoded protein by changing only the nucleotides that have been characterized as essential in recruiting the selenocysteine incorporation machinery in E. coli.

In order to design an E. coli SECIS element the SECISdesign server (http://www.bioinf.uni-freiburg.de/Software/SECISDesign/) was used to design SECTS-elements within the coding sequence. The design was based on the natural SECIS-element FdhF of E. coli with all bonds of the stem-loop structure maintained.

The resulted sequence where the native Moorella SECTS element was substituted with the FdhF-based E. coli FDH is shown in FIG. 39A (SEQ ID NO: 8).

The SECIS element substitution resulted in a modified ORF with two amino-acid changes (underlined) in the encoded protein (Table 5).

The hybrid cDNA was further codon optimized to achieve higher expression levels in E. coli, except of the sequence coding for the E. coli SECIS element. Codon optimization was done for the Moorella FDH-B. Therefore, the final synthesized sequences by Genscript are shown in FIG. 39B (SEQ ID NO: 9) and FIG. 39C (SEQ ID NO: 10).

Both FDH-A and FDH-B optimized were subcloned into the pETDuet-1 vector that facilitates the co-expression of two target genes. We developed in vivo assays to assess the in vivo formaldehyde dehydrogenase and formate dehydrogenase activity in their reverse direction. The in vivo assay designed for this purpose is as follows:

(a) Start culture and incubate at 37° C. overnight in LB media. Inoculate fresh TB media and incubate until OD₆₀₀=1-2;

(b) Spin down the bacteria at 4000 g for 10 min at 4° C.;

(c) Resuspend bacterial pellet in minimal media (M9, MOPS) supplemented with glucose or glycerol;

(d) Induce with 0.2-1 mM IPTG and transfer the culture into serum bottles to be cultivated anaerobically;

(e) After 1-2 hours of incubation at 37° C. add 1 mM methyl-viologen to maintain the media reduced;

(f) Addition of sodium bicarbonate to provide the substrate for the in vivo activity; and

(g) Let the culture grow at 37° C., sample at 1-18 h and test for protein expression, formate and formaldehyde concentration.

The formaldehyde dehydrogenase and formate dehydrogenase engineered strains were transformed with an extra plasmid carrying the Nostoc sp carbonic anhydrase that is expected to allow conversion of the HCO₃ ⁻ to CO₂, the substrate for the formate dehydrogenase in the reverse direction.

Scheme 2. The reverse tricarboxylic acid (rTCA) cycle (FIG. 40) is one of 6 autotrophic CO₂ fixation cycles found in nature. rTCA is similar to the oxidative TCA cycle in E. coli; however, in the cycle reversal, molecules of CO₂ are fixed using reducing equivalents and ATP to produce acetyl-CoA. Many steps in the TCA cycle are reversible under standard conditions, including the aconitase, succinyl-CoA synthase, fumarase, and malate DH reactions. In most organisms using the rTCA cycle, there are 4 reactions for which alternative enzymes are needed to drive the reverse reaction towards CO₂ fixation: ATP citrate lyase (ACL), 2-oxoglutarate: ferredoxin oxidoreductase (OGOR), isocitrate DH (ICDH), and fumarate reductase (FR).

Scheme 3. The glycine synthase is a reversible mechanism of CO₂ fixation used by a number of clostridia when metabolizing reduced substrates. The glycine cleavage system begins by reducing CO₂ to formate using FDH. Formate is bound to a tetrahydrofolate (THF) coenzyme by formate-THF ligase in an ATP-dependent reaction. The resulting 10-formyltetrahydrofolate is reduced to 5,10-methylene-THF at which point the methylene-group is condensed with CO₂ and NH₃ to form glycine. In some bacterial species, the glycine can be converted to acetyl-Pi and then dephosphorylated. An alternate route uses an additional moiety of methylene-THF to produce serine, which can then be deaminated to form pyruvate. A large number of these reactions are native to E. coli. The 4 remaining enzymes that will be needed are aminomethyltransferase (AMT), dehydrolipoyl dehydrogenase (LPDH), glycine dehydrogenase (GDH), plus a CO₂-reducing FDH.

Example 27

Metabolic Engineering to Enable E. coli to Grow Effectively on MeOH

Methanol will be used as a carbon source by conversion to HCHO by a MeOH dehydrogenase (MDH). HCHO will then be converted to hexulose-6-phosphate, using ribulose-5-phosphate, by a hexulose phosphate synthase (HPS). Hexulose phosphate isomerase (PHI) will convert the hexulose-6-phosphate to fructose-6-phosphate, which can then be used for pyruvate generation that can be fed into the n-BuOH producing pathway (FIG. 2). There is strong experimental evidence supporting functionality of these genes, individually, in E. coli.

The methanol dehydrogenase (mdh) enzymes from Bacillus stearothermophilus (mdh1) and Bacillus methanolicus PB1 (mdh2) as well as an activator protein (act) from B. methanolicus PB1 were cloned into the pETM6 expression vector. MDH allows the cells to use methanol as a growth substrate, and the activator protein stimulates activity of the MDH of B. methanolicus PB1. The enzyme activity assay protocol of Krog et al. (PLOS One. 2013; 8(3):59188) was adapted to measure the K_(m) and specific activity for each enzyme. Specific activities and K_(m) values for MDH1 (from B. stearothermophilus), MDH2 (from B. methanolicus PB1), and MDH2 with ACT (the activator protein from B. methanolicus), were determined in crude lysates (FIG. 41). K_(m) values for methanol were determined as 27 mM for MDH and 101 mM for MDH2, similar to those reported previously. Specific activities for MDH and MDH2 were determined as 0.014 and 0.008 μmol/min/mg, respectively, in crude cell extracts. The ACT of B. methanolicus PB1 also stimulated MDH2 activity in vitro, yielding a K_(m) of 119 mM and a specific activity of 0.009 μmol/min/mg. Cloning of several other reported methanol dehydrogenases is in progress to identify the best candidates to move forward with.

Primers were designed to amplify the Mycobacterium gastri phi-hps operon, which encodes the genes necessary to fix formaldehyde to ribulose-5-phosphate yielding hexulose-6-phosphate, and then convert ribulose-5-phosphate to fructose-6-phosphate. Genes were amplified and initially cloned into the expression vector pET21a (Novagen). This vector utilizes the T7 promoter, and was transformed into the Rosetta strain of E. coli. E. coli Rosetta expressing the M. gastri PHI-HPS operon was compared with an empty vector control for growth in LB with 2 mM formaldehyde (FIG. 42A-B). E. coli cells expressing the phi and hps genes from M. gastri were able to grow in LB plus formaldehyde (FIG. 42A) or a mixture of M9 and LB plus formaldehyde (FIG. 42B), whereas cells containing only an empty vector control were unable to grow under either of the conditions tested. When the same strains were tested in M9 minimal media containing only formaldehyde (0.25 mM) as the sole carbon source, no increase in OD₆₀₀ was observed (FIG. 43). However, the strain expressing the phi and hps genes was able to consume the formaldehyde at a faster rate than the empty vector control strain, indicating that the genes were functional but there was most likely not enough of the carbon source to support meaningful growth of the culture.

We were able to incorporate the B. stearothermophilus_mdh gene into the vector containing the M. gastri phi-hps operon (pET_M.g._PHI_HPS) and successfully transform E. coli with the resulting three gene plasmid. However, we failed to detect MDH activity in culture lysates. As such, we have abandoned the use of the pET21 vector (Novagen) given its limitations in incorporating multiple gene pathways and have begun working with the pETM6 vector. This vector is designed for the sequential addition of genes in a multi-gene pathway and allows for each gene to be under the control of its own promoter. Therefore, we have decided to use this expression system henceforth for cloning and expressing the mdh and RuMP genes as this will allow us to combine different genes from different organisms and determine which combination gives the best growth rates.

Additionally, the genome of the methylophic bacteria strain L3 has been sequenced to provide for additional candidate genes for methanol utilization. We found that the L3 strain contains two putative hps genes and one putative phi gene, all of which are currently being cloned into the pETM6 vector in order to compare their abilities for formaldehyde consumption with the genes from M. gastri.

Example 28

Metabolic Engineering to Enable E. coli to Fix CO₂ when Growing on MeOH

To achieve this, we will explore two schemes and possibly a third one as a backup. First, for all schemes, we will overexpress a carbonic anhydrase (CA) in order to increase the availability of CO₂ to the cells and drive the overall reaction to HCHO formation. CA catalyzes the hydration of CO₂ to HCO₃ ⁻ and plays an important role in increasing the effective CO₂ concentration for CO₂ fixation in cyanobacteria. CA from a cyanobacterium (Anabaena sp. 7120) was shown to enhance CO₂ utilization in E. coli, and thus we propose to use the same gene in our strains. We note that the physiological role of this CA is to enhance CO₂ uptake, unlike the role of the native E. coli CA, which is part of an operon induced under cyanate stress and thus has likely an opposite role. Nevertheless, the native CA did not interfere with the beneficial effect of the recombinant CA. Based on this discussion, we will co-express CA with formate DH (FDH) and HCHO DH (FdDH) as a first step toward enhancing the overall CO₂ fixation. A similar strategy coupling the use of CA with a 3-enzyme pathway improved the formation of MeOH from CO₂ by >4-fold. CO₂ fixation is best and most easily assessed by following the fate of ¹³CO₂ or ¹³C-bicarbonate by GC-MS.

SCHEME 1: CO₂ reduction to formate and HCHO will use a FDH and a FdDH (or a HCHO dismutase (FdDM)), respectively (FIG. 2 & Table 6). One may be concerned with the thermodynamic feasibility of using FDH and FdDH to enable CO₂ capture. Indeed, under most circumstances the preferred reaction direction oxidizes CH₂O to CO₂. Yet, there are a number of conditions both in vivo and in vitro, in which the reduction of CO₂ to HCHO is possible, despite the computed positive Δ_(r)G′ numbers [calculations used the eQuilibrator with 0.1 M ionic strength, pH=7, and reactants/products at 1 mM]: CO₂+NADPH→HCOOH+H₂O+NADP⁺ (Δ_(r)G′=13.2 kJ/mol*) HCOOH+NADPH→HCHO+H₂O+NAD⁺ (Δ_(r)G′=44.2 kJ/mol*)

Indeed, in vivo, reduction of CO₂ to formate (some at very high rates) using an NADPH-dependent FDH is widespread in the CO₂-fixing acetogens, which use the Wood-Ljungdahl pathway (WLP). Acetogen FDHs favor the reduction of CO₂ to formate due to the tight binding of NADPH to the enzyme, and this was recently shown to be the case in vitro as well for the enzyme we plan to use. Substrate binding and other micro-environmental conditions dramatically change the local concentrations in the gel-like cellular milieu (as compared to the standard solution environments used for calculations) to make this reaction possible. Biological reduction of formate to HCHO has also been demonstrated, both in vivo and in vitro. Enzyme studies have shown the use of an FdDH to produce HCHO from formate using NADH or reduced viologen. In vitro, this reaction was also clearly demonstrated using a yeast FdDH, which requires glutathione (GSH; a tripeptide produced natively at good rates in E. coli). An alternate route to produce HCHO from formate is the reverse HCHO dismutase reaction [HCOOH+MeOH

2 (HCHO)+H₂O] using MeOH as an electron source, rather than NAD(P)H. This reaction is observed in methylotrophs that lack a dedicated MDH, which necessitates the presence of both HCOOH and MeOH for growth.

The open reading frames of two FDH genes, three FdDH genes and one CA gene were codon optimized for E. coli expression and cloned into pET (Novagen) expression vectors (Table 7). The fdh genes are from Clostridium carboxidivorans and Eubacterium acidaminophilum, and the ca is from a Nostoc species. The fld genes originate from Rhodobacter sphaeroides, Methylobacillus flagellatus, and Candida boindini. Crude protein lysates of over-expressing E. coli strains expressing C. boindini FLD and R. sphaeroides FLD (FIG. 44A) were tested for activity in vitro by monitoring NADH production in the reaction: formaldehyde+H₂O+NAD⁺

formate+NADH. The C. boindini and the R. sphaeroides were found active in both directions (FIG. 44B-D). M. flagellatus enzyme was not expressed at visible levels and when tested the clear lysate was found inactive (FIG. 44D).

Example 29

Engineering E. coli to Grow Well on MeOH and Fix CO₂

We desire to engineer E. coli to grow well on MeOH and fix CO₂ driven by the excess reduction energy derived from MeOH use under anaerobic conditions. To achieve this, we must assemble the best possible genes/enzymes for the 3 modules (MeOH use; CO₂ fixation; n-BuOH formation; FIG. 2). Each of these goals must be carried out efficiently and assessed for effectiveness of outcomes in terms of rates of MeOH and CO₂ utilization and carbon fluxes to pyruvate, acetyl-CoA and n-BuOH (FIG. 2).

1. Strain Engineering

The proposed approach is to simultaneously use MeOH and CO₂ to produce n-butanol (n-BuOH) (FIG. 2). This will be achieved by leveraging genes from methylotrophs for MeOH utilization (Module 1); various genes from acetogens and other organisms for CO₂ fixation (Module 2), and these two modules will be combined with the n-BuOH Module 3 that has already been constructed and demonstrated for efficiency.

2.a. Genes for Metabolic Engineering to Enable E. coli to Grow Effectively on MeOH.

Methanol will be used as a carbon source by conversion to HCHO by a MeOH dehydrogenase (MDH). HCHO will then be converted to hexulose-6-phosphate, using ribulose-5-phosphate, by a hexulose phosphate synthase (HPS). Hexulose phosphate isomerase (PHI) will convert the hexulose-6-phosphate to fructose-6-phosphate, which can then be used for pyruvate generation that can be fed into the n-BuOH producing pathway (FIG. 2). There is strong experimental evidence supporting functionality of these genes, individually, in E. coli.

The mdh gene of Bacillus methanolicus C1 was cloned in E. coli and used for protein purification. While we will first use the B. methanolicus C1 mdh, in case this gene is not effective, we have compiled a list of alternative genes (Table 8) that we can use including the MDH from Methylobacterium extorquens AM1. The recent genome sequences of B. methanolicus strains MGA3 and PB1 revealed that there are three different mdh genes in each of the two strains. The 3 B. methanolicus MGA3 mdh genes are quite different, with mdh1 sharing 22% and 60% homology with mdh2 and mdh3. The mdh2 and mdh3 genes share only 22% homology. However, on the protein level, Mdh2 and Mdh3 are 96% identical, and share 61% and 62% sequence homology with Mdh. We will initially clone mdh and investigate whether the resulting protein is functional in E. coli. If the activity is low, we will clone the additional proteins on a single plasmid. Another option is the alcohol DH (ADH) from B. stearothermophilus, which was cloned into E. coli and whereby oxidation of methanol was demonstrated. NAD+ was used as a cofactor for methanol oxidation. NAD/NADH is the most desirable coenzyme for this reaction as NADH produced from this reaction will be used to drive CO₂ fixation and BuOH production.

The next two enzymes, HPS and HPI, have also been successfully cloned into E. coli. Cell extracts of E. coli containing an expression plasmid with the Bacillus subtilis hps and phi genes were used to show good activity for these enzymes. Furthermore, ¹³C NMR demonstrated that HCHO was incorporated into hexulose-6-phosphate and fructose-6-phosphate. In addition, the hps and phi cluster from B. brevis S1 was cloned into E. coli to demonstrate good HPS and PHI activities. More recently, a gene coding for an HPS-PHI fusion from Mycobacterium gastri MB19 was expressed in E. coli and cells were able to metabolize HCHO added to the culture and continue growing. We have also included the Methylococcus capsulatus MCA2738 gene that is annotated to have HPS and PHI activities, as another option to investigate. Combined, these data strongly suggest that expression of MDH, HPS and PHI expression in E. coli is feasible and can facilitate the formation of F6P from MeOH through HCHO via the RuMP pathway. We will first express these genes alone, test in vitro activities, choose the genes that lead to the two highest activities and then express these genes combinatorially aiming to identify at least 2 combinations of the 3 genes that give the best growth on MeOH prior to pursuing Tasks 3 and 4. Effectiveness is assessed by the rate of growth on MeOH and final densities (by OD₆₀₀ measurements), as well as rates of MeOH utilization (by GC and/or HPLC). We will also examine metabolites (acetate, ethanol, higher carboxylic acids; by HPLC) that will likely be produced anaerobically until we engineer this module into the n-BuOH producing strain. Strategies for expressing these genes are discussed below. The Gibson assembly method enables quick plasmid construction for screening to identify the best genes and the best combinations.

2.b. Metabolic Engineering to Enable E. coli to Fix CO₂ when Growing on MeOH

To achieve this, we will explore two schemes and possibly a third one as a backup. First, for all schemes, we will overexpress a carbonic anhydrase (CA) in order to increase the availability of CO₂ to the cells and drive the overall reaction to HCHO formation. CA catalyzes the hydration of CO₂ to HCO₃ ⁻ and plays an important role in increasing the effective CO₂ concentration for CO₂ fixation in cyanobacteria. CA from a cyanobacterium (Anabaena sp. 7120) was shown to enhance CO₂ utilization in E. coli, and thus we propose to use the same gene in our strains. We note that the physiological role of this CA is to enhance CO₂ uptake, unlike the role of the native E. coli CA, which is part of an operon induced under cyanate stress and thus has likely an opposite role. Nevertheless, the native CA did not interfere with the beneficial effect of the recombinant CA. Based on this discussion, we will co-express CA with formate DH (FDH) and HCHO DH (FdDH) as a first step toward enhancing the overall CO₂ fixation. A similar strategy coupling the use of CA with a 3-enzyme pathway improved the formation of MeOH from CO₂ by >4-fold. CO₂ fixation is best and most easily assessed by following the fate of ¹³CO₂ or ¹³C-bicarbonate by GC-MS.

SCHEME 1: CO₂ reduction to formate and HCHO will use a FDH and a FdDH (or a HCHO dismutase (FdDM)), respectively (FIG. 2 & Table 9). One may be concerned with the thermodynamic feasibility of using FDH and FdDH to enable CO₂ capture. Indeed, under most circumstances the preferred reaction direction oxidizes CH₂O to CO₂. Yet, there are a number of conditions both in vivo and in vitro, in which the reduction of CO₂ to HCHO is possible, despite the computed positive Δ_(r)G′ numbers [calculations used the eQuilibrator with 0.1 M ionic strength, pH=7, and reactants/products at 1 mM]: CO₂+NADPH→HCOOH+H₂O+NADP⁺ (Δ_(r)G′=13.2 kJ/mol*) HCOOH+NADPH→HCHO+H₂O+NAD⁺ (Δ_(r)G′=44.2 kJ/mol*)

Indeed, in vivo, reduction of CO₂ to formate (some at very high rates) using an NADPH-dependent FDH is widespread in the CO₂-fixing acetogens, which use the Wood-Ljungdahl pathway (WLP). Acetogen FDHs favor the reduction of CO₂ to formate due to the tight binding of NADPH to the enzyme, and this was recently shown to be the case in vitro as well for the enzyme we plan to use. Substrate binding and other micro-environmental conditions dramatically change the local concentrations in the gel-like cellular milieu (as compared to the standard solution environments used for calculations) to make this reaction possible. Biological reduction of formate to HCHO has also been demonstrated, both in vivo and in vitro. Enzyme studies have shown the use of an FdDH to produce HCHO from formate using NADH or reduced viologen. In vitro, this reaction was also clearly demonstrated using a yeast FdDH, which requires glutathione (GSH; a tripeptide produced natively at good rates in E. coli). An alternate route to produce HCHO from formate is the reverse HCHO dismutase reaction [HCOOH+MeOH

2 (HCHO)+H₂O] using MeOH as an electron source, rather than NAD(P)H. This reaction is observed in methylotrophs that lack a dedicated MDH, which necessitates the presence of both HCOOH and MeOH for growth.

SCHEME 2: The reverse tricarboxylic acid (rTCA) cycle (FIG. 40) is one of 6 autotrophic CO₂ fixation cycles found in nature. rTCA is similar to the oxidative TCA cycle in E. coli; however, in the cycle reversal, molecules of CO₂ are fixed using reducing equivalents and ATP to produce acetyl-CoA. Many steps in the TCA cycle are reversible under standard conditions, including the aconitase, succinyl-CoA synthase, fumarase, and malate DH reactions. In most organisms using the rTCA cycle, there are 4 reactions for which alternative enzymes are needed to drive the reverse reaction towards CO₂ fixation: ATP citrate lyase (ACL), 2-oxoglutarate: ferredoxin oxidoreductase (OGOR), isocitrate DH (ICDH), and fumarate reductase (FR) (Table 10)

ACL uses ATP hydrolysis to drive the unfavorable thermodynamics towards the formation of oxaloacetate and acetyl-CoA. The ac/BA genes from Chlorobium tepidum and C. limicola have been functionally expressed in E. coli, and either set of genes would be suitable for the goal here. OGOR catalyzes the carboxylation of succinyl-CoA to form α-ketoglutarate. Two distinct enzymes have been identified in autotrophs with the rTCA cycle: For and Kor. These enzymes have been best studied in the H₂-oxidizing bacterium Hydrogenobacter thermophilus, which grows quickly on H₂, CO₂, and O₂ with a doubling time of ˜1 h. For consists of 5 subunits that are encoded in the forDABGE gene cluster and is necessary for aerobic growth. Kor has 2 subunits coded on the korAB gene cluster and is essential for anaerobic growth; this will be used here. Both enzymes use reduced ferredoxin, which has a greater reduction potential than NADH, to drive the endergonic carboxylation of succinyl-CoA. Organisms that use the rTCA cycle have specialized ICDH enzymes that preferentially operate in the carboxylating direction. ICDH from C. limicola, for instance, has the same affinity for carboxylation as for decarboxylation at physiological pH, as compared to ICDH from other organisms, in which decarboxylation is favored 4:1. ICDH activity in H. termophilus operates through an entirely different mechanism than that of C. limicola or the oxidative TCA cycle. H. thermophilus has 2 enzymes—2-oxoglutarate carboxylase (OGC) and oxalosuccinate reductase (OSR)—that carboxylate α-ketoglutarate in two steps. First, OGC uses ATP to carboxylate α-ketoglutarate to oxalosuccinate, and OSR then converts oxalosuccinate to isocitrate. FR catalyzes the reduction of fumarate to succinate. Since E. coli already makes use of fumarate reductase for anaerobic respiration, overexpression of heterologous genes may not be necessary.

SCHEME 3: The glycine synthase is a reversible mechanism of CO₂ fixation used by a number of clostridia when metabolizing reduced substrates. The glycine cleavage system begins by reducing CO₂ to formate using FDH. Formate is bound to a tetrahydrofolate (THF) coenzyme by formate-THF ligase in an ATP-dependent reaction. The resulting 10-formyltetrahydrofolate is reduced to 5,10-methylene-THF at which point the methylene-group is condensed with CO₂ and NH₃ to form glycine. In some bacterial species, the glycine can be converted to acetyl-P^(i) and then dephosphorylated⁵³. An alternate route uses an additional moiety of methylene-THF to produce serine, which can then be deaminated to form pyruvate. A large number of these reactions are native to E. coli. The 4 remaining enzymes that will be needed are aminomethyltransferase (AMT), dehydrolipoyl dehydrogenase (LPDH), glycine dehydrogenase (GDH), plus a CO₂-reducing FDH.

2.c. Expression Details. Protein and Enzymatic Assays

Expression vectors and promoters. The genes listed in Tables 7-9 are to be cloned in two compatible plasmids. The genes associated with MeOH consumption, mdh, hps and phi, totaling approximately 2700 bp, will be cloned on a plasmid with the p15A origin of replication. The genes enabling CO₂ utilization (ecaA, fdh, and fddh, or alternates as above) will be cloned on a high copy pBR322 origin of replication plasmid. All genes will be first cloned under the strong tac promoter, with rho independent terminators between each gene. The genes will be codon optimized for E. coli, and cloning will be done via Gibson assembly. After cloning, genes will be evaluated for good expression by qRT-PCR.

Enzyme assays and Western blots to ensure that functional proteins are produced. We will first employ functional enzymes assays as detailed below. Once we settle on a smaller set of genes; we will generate antibodies against the corresponding proteins. We will use a commercial vendor that has successfully generated many antibodies for a large variety of proteins for other projects. The antibodies will be used to carry out Western blots in order to quickly assess the impact of optimized expression and culture conditions on the protein levels of these recombinant proteins. Enzyme assays will be carried out for the reactions to enable MeOH utilization and CO₂ fixation. The main focus of this effort is on the MeOH utilization pathway and the CO₂ fixation through formate and CH₂O. The presence of the native TCA cycle enzymes would complicate in vitro assays of the rTCA cycle SCHEME 2. For that and the backup SCHEME 3, we will rely on mRNA levels of expression by Q-RT-PCR. MDH, FdDH and FDH activity can be also monitored using alternate assays. PHI activity will be also assayed.

2.d. Integrate Optimized Modules 1, 2 and 3 (FIG. 2) to Achieve Effective MeOH Utilization, CO2 Fixation and n-BuOH Formation.

The assembled Modules 1 and 2 (FIG. 2) will be optimized to achieve high rates of MeOH and CO2 utilization and n-BuOH formation. The goal is to integrate and further optimize these pathways by combing the best of these into a single strain that does not produce BuOH and later after some testing, into a BuOH producing strain. We will then employ chemical mutagenesis and strain evolution to achieve better growth on MeOH, CO₂ fixation and n-BuOH production. BuOH producing strain contains several gene deletions aiming to enhance the electron availability for BuOH production under anaerobic conditions. The strain then expresses a clostridial pathway for n-BuOH, with the genes expressed from co-existing plasmids. With the additional genes (FIG. 2) that will be needed here, we will need to integrate several of the genes from Modules 1 and 3 (FIG. 2) and possibly from the BuOH-formation pathway into the chromosome. What will be integrated will be based on mRNA expression levels that we will measure by qRT-PCR. We have employed chromosomal integrations of multiple genes into the E. coli genome using the lambda-red system, so we will use these methods for chromosomal integration of genes. Strains that combine all 3 modules (FIG. 2) will be tested for growth on MeOH, CO₂ fixation and BuOH production. A few of the best performing strains will be evolved through chemical mutagenesis and fast transfer for faster growth on MeOH anaerobically under a CO₂ and also H₂ atmosphere (3 atm) is serum vials used to grow acetogens on gas mixtures of CO₂/CO/H₂. The CO₂ and H₂ pressure is to stimulate CO₂ uptake under a reducing environment and is meant to simulate the large-scale process where a large CO₂ pressure will be built during the anaerobic fermentation. A few of the best performing strains with gene integrations and accumulated mutations will be sequenced by the new PacBio technology. The goal will be to examine the mutations that improve the phenotype.

Example 30

Bioenergetic and Pathway Analysis

1. Conversion of Methanol to Acetyl-CoA and Butanol

We will engineer E. coli cells to utilize MeOH as a carbon and energy source in an engineered RuMP pathway. To meet ARPA-E yield and energy efficiency we will use CO₂ recycling by Schemes 1 or 2 (or 3 if necessary) (FIG. 2). In Scheme 1, CO₂ is reduced to formate and then HCHO (rxn 2 of FIG. 2) that can be assimilated through the RuMP pathway. Scheme 2 utilizes the rTCA cycle (FIG. 40). Backup Scheme 3 uses the glycine cleavage pathway via the glycine synthase. While Schemes 2 and 3 are not as ATP efficient (see rxn 3 of FIG. 2) as Scheme 1, ATP hydrolysis improves the overall thermodynamic outlook of CO₂ fixation. Maximum theoretical yields were calculated per ARPA-E instructions for the engineered RuMP pathway with CO₂ recycling through either Scheme 1, 2 or 3 (Table 11). In both cases (Scheme 1; Scheme 2 or 3), the calculated yields outperform the energy efficiency and carbon yield metrics of >64% and >67%, respectively, even if we assume that 80% (our target) of theoretically possible CO₂ is fixed (in fact ca. 67% of the theoretically possible CO₂ fixation would suffice).

2. The Rate of Product Formation

For producing n-BuOH, we will employ the recently reported system, which produces ca. 30 g/L n-BuOH, but at a rate <1 g/gCDW/h. To meet the latter rate, we will need to optimize all 3 modules (FIG. 2). We will first aim to achieve fluxes to acetyl-CoA that supports high fluxes of product formation for products synthesized through Acetyl-CoA. Then, we will assess the bottlenecks in Module 3 (BuOH synthesis) by relating flux data to mRNA levels of key genes to examine if gene expression of certain genes is limiting the flux. Based on flux data, mRNA data and enzyme assays, we will identify the steps that limit the overall rate, and will use an iterative optimization process to achieve the most efficient strain and process.

3. Technological Scenarios for Conversion of CH₄ to BuOH

The four possible outcomes CH4 activation are shown in FIG. 1, with case III representing the status quo with two electrons lost, case IV representing all electrons conserved for CH₄ to MeOH conversion, and cases II and III representing intermediate levels of electron loss. The conservation of electrons in the CH₄ activation has a dramatic impact on product yields as shown in Table 12. The status quo of CH₄ activation (Case III), results in the lowest calculated BuOH yield which will not likely find industrial applications. In contrast, if all electrons are conserved (Case IV), an abundance of reducing energy allows for net CO₂ fixation using Scheme 1 for CO₂ recycling and 100% carbon efficiency with respect to CH₄ under Schemes 2 or 3.

TABLE 1 NAD⁺-dependent MDHs. Source MDH Bacillus stearothermophilus 2334 Mdh Bacillus stearothermophilus NUB3621 Mdh Bacillus methanolicus C1 Mdh Bacillus methanolicus PB1 Mdh Mdh1 Mdh2 Bacillus methanolicus MGA3 Mdh Mdh2 Mdh3 Table 2. Reaction Gibbs energies (Δ_(r)G), equilibrium constants (K_(eq)), and change in reduction potential (Δε) for MeOH redox reactions calculated through eQuilibrator software. Standard conditions were 25° C., 1 bar, pH 7.0, 0.1 M ionic strength, and 1 mM species concentrations. Physiological conditions were the indicated temperatures, 1 bar, pH 7.6, 0.1 M ionic strength, 1250 mM MeOH, 0.17 mM HCHO, 2.6 mM NAD, 0.083 mM NADH. Data were calculated from the following equations: Δ_(r)G=−n(23,064)Δε, where n represents the number of electrons transferred, K_(eq)=10^(−Δ) ^(r) ^(G′/2.3RT), and

${{\Delta_{r}G} = {{\Delta_{r}G^{\prime}} + {{RT}\;\ln\frac{\lbrack P\rbrack}{\lbrack R\rbrack}}}},$ where P and R indicate products and reactants, respectively. ^(a) Values in parentheses under physiological conditions were calculated with a 10-fold decrease in HCHO concentration, i.e., 0.017 mM. The discrepancy between some of the Δ_(r)G and K_(eq) values for NAD-dependent oxidation is likely a result of the 95% confidence interval for Δ_(r)G, which is ±6.5 kJ/mol.

Methanol Standard Conditions Physiological Conditions Redox Δε′ Δ_(r)G′ T Δ_(r)G Reaction (mV) (kJ/mol) K′_(eq) (° C.) Δε (mV) (kJ/mol) K_(eq) CH₃OH + −177 +34.2 1.0 × 37  +5.3 −1.0 0.925 NAD  

10⁻⁶ (+36.1)^(a) (−7.0)^(a) (8.5)^(a) HCHO + 45  +9.6 −1.8 0.927 NADH + H⁺ (+41.1)^(a) (−7.9)^(a) (8)^(a)   55 +14.9 −2.9 0.929 (+47.4)^(a) (−9.2)^(a) (7.5)^(a)

TABLE 3 Peptide encoded by the artificial SECIS element  compared to the peptide encoded by the original  sequence that follows the selenocysteine. Original sequence H S S T V A G L A T T F G S  (starting at (SEQ ID NO: 4) position 359) Optimized amino- H S S T V A G L  H Q  T F G S acid sequence (SEQ ID NO: 5)

TABLE 4 NAD⁺-dependent MDH candidates MDH Codon Optimized in vivo Activity Bst 2334 Mdh No Yes Bst NUB3621 Mdh Yes Yes Bme C1 Mdh Yes No Bme PB1 Mdh Yes No Bme PB1 Mdh1 Yes No Bme PB1 Mdh2 Yes No Bme MGA3 Mdh Yes No Bme MGA3 Mdh2 Yes Yes Bme MGA3 Mdh3 Yes Yes

TABLE 5 Peptide encoded by the artificial SECIS element compared to the peptide encoded by the original sequence that follows the selenocysteine. Original Sequence H S S T V A G L A T T F G S  (starting at (SEQ ID NO: 4) pos. 359): Amino Acid Sequence H S S T V A G L  L A  T F G S (SEQ ID NO: 11)

TABLE 6 CO₂ fixation genes via SCHEME 1 Gene Source Organism Size (bp) Kinetics CA (ecaA) Nostoc sp. PCC 7120 795 Activity: 21.8 (Anabaena sp. PCC7120) U/mg protein FDH (fdh) Clostridium carboxidivorans 2142 Km = 0.05 mM P7^(T) (for NADH) FdDH (fddh1) Candida boidinii Fld1 2490 N/A HCHO DM Pseudomonas putida 9816 1197 N/A

TABLE 7 List of protein-expression constructs to be used in SCHEME 1 of the CO₂ fixation module. Vector Codon Construct (antibiotic optimization name resistance) Insert for E. coli His-tag Sequenced  1 PET21a:opt pET21a(+) Rhodobacter YES NO YES Rhodo_FLD (amp) sphaeroides FLD (Genscript)  2 PET21a:opt pET21a(+) Methylobacillus YES NO YES Methylo_FLD (amp) flagellatus FLD (Genscript)  3 PET21a:DNA2 pET21a(+) Candida boindini YES NO YES CanBo_FLD (amp) FLD (DNA2.0)  4 PET21a:Gen pET21a(+) Candida boindini YES NO YES CanBo_FLD (amp) FLD (Genscript)  5 PDUET1:opt pETDuet-1 Rhodobacter YES YES In Rhodo_FLD (amp) sphaeroides FLD (Genscript) N- progress terminal  6 PDUET1:opt pETDuet-1 Methylobacillus YES YES In Methylo_FLD (amp) flagellatus FLD (Genscript) N- progress terminal  7 PET21a:opt pET21a(+) Clostridium YES YES YES CloCa_FDH (amp) carboxidivorans (IDT) C- FDH² terminal  8 PACYC:opt pACYCDuet-1 Clostridium YES NO YES CloCa_FDH carboxidivorans (IDT) FDH  9 PACYC:opt pACYCDuet-1 Eubacterium YES NO In Euacid_FDH acidaminophilum (Genscript) progress FDH-II 10 PET28A:opt pET28a(+) Clostridium YES YES YES CloCa_FDH carboxidivorans (IDT) N- FDH terminal 11 PET28A:opt pET28a(+) Eubacterium YES YES In Euacid_FDH acidaminophilum (Genscript) N- progress FDH-II terminal 12 PET21a:CA pET21a(+) Nostoc sp YES NO YES (amp) carbonic (Genscript) anhydrase 13 PET21a:CA pET21a(+) Nostoc sp YES NO YES (amp) carbonic (DNA2.0) anhydrase

TABLE 8 Genes to enable growth of E. coli on MeOH with enzyme characteristics when known Kinetics (BRENDA): K_(m) (mM) Gene Spec. act. Gene Organisms source size (bp) [μmol/min/mg] mdh Bacillus methanolicus C1 1149 0.02, 1.2 Bacillus stearothermophilus 1471 Bacillus methanolicus MGA3 1149, mdh, mdh2, mdh3 1158, 1158 Methylobacterium extorquens 1845 AM1 mxaF (MDH subunit alpha) 291 mxaI (MDH subunit beta) hps Bacillus subtilis 633 0.007 (Bacillus sp.), 3.5 Bacillus brevis S1 633 N/A, 7.4 Methylococcus capsulatus 1170 MCA2738 (HPS and PHI activity) Mycobacterium gastri MB19 624 phi Bacillus subtilis 558 N/A, 11 Bacillus brevis S1 552 Methylococcus capsulatus 1170 MCA2738 (HPS and PHI activity) Mycobacterium gastri MB19 600

TABLE 9 CO₂ fixation genes via SCHEME 1 Gene Source Organism Size (bp) Kinetics CA (ecaA) Nostoc sp. PCC 7120 795 Activity: 21.8 (Anabaena sp. PCC7120) U/mg protein FDH (fdh) Clostridium carboxidivorans 2142 Km = 0.05 mM P7^(T) (for NADH) FdDH (fddh1) Candida boidinii Fld1 2490 N/A HCHO DM Pseudomonas putida 9816 1197 N/A

TABLE 10 Enzymes and genes for the rTCA cycle Enzymes Source Organism Genes ATP Citrate Lyase (ACL) C. tepidum or C. limicola aclA, aclB OGOR (For and Kor) H. thermophilus korAB ICDH C. limcola or H. thermophilus idh Fumarate Reductase (FR) E. coli or H. thermophilus frdABCDE

TABLE 11 n-BuOH from MeOH (mol/100 mol MeOH) CO₂ fixation Scheme 1 Scheme 2 or 3 Methanol −100.0  −100.0  CO2  0.0  0.0 NAD(P)H  0.0  0.0 ATP 50.0  0.0 Butanol 25.0 25.0 Energy Efficiency 96% 96% Carbon Yield 100%  100% 

TABLE 12 n-BuOH from CH₄ (mol/100 mol CH₄). CO₂ fixation CH₄ Act. Scheme 1 Scheme 2 or 3 Scheme* I II III^(#) IV I II III^(#) IV Methane −100.0 −100.0 −100.0 −100.0 −100.0 −100.0 −100.0 −100.0 CO₂ 0.0 16.7 33.3 −33.3 0.0 16.7 33.3 0.0 NAD(P)H 0.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 ATP 50.0 41.7 33.3 66.7 0.0 16.7 33.3 0.0 Butanol 25.0 20.8 16.7 33.3 25.0 20.8 16.7 25.0 Energy  77% 64% 51% 102%  77% 64% 51%  77% Efficiency Carbon 100% 83% 67% 133% 100% 83% 67% 100% Yield *See FIG. 1 and text for CH₄ activation schemes.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims. 

What is claimed:
 1. A method for producing a metabolite, comprising (a) growing a non-naturally occurring microbe in a medium comprising methanol, wherein the non-naturally occurring microbe expresses heterologous Bacillus stearothermophilus methanol dehydrogenase (MDH) and one or more heterologous ribulose monophosphate (RuMP) pathway enzymes, wherein the one or more heterologous RuMP pathway enzymes consist of Bacillus methanolicus 3-hexulose-6-phosphate synthase (HPS) and Bacillus methanolicus 3-hexulose-6-phosphate isomerase (PHI), and wherein the non-naturally occurring microbe is Escherichia coli, (b) producing a metabolite by the non-naturally occurring microbe, and (c) incorporating carbon from methanol into the metabolite, wherein at least 40% of the carbon in the metabolite is derived from the methanol.
 2. The method of claim 1, wherein the expression of the one or more heterologous RuMP pathway enzymes is under the control of a formaldehyde responsive promoter.
 3. The method of claim 1, wherein the non-naturally occurring microbe contains a deletion of a frmRAB operon.
 4. The method of claim 1, wherein the non-naturally occurring microbe further expresses one or more heterologous pentose-phosphate pathway (PPP) enzymes.
 5. The method of claim 4, wherein the expression of the one or more heterologous PPP enzymes is under the control of a formaldehyde responsive promoter.
 6. The method of claim 4, wherein the one or more heterologous PPP enzymes consist of heterologous phosphofructokinase (PFK), heterologous fructose bisphosphate aldolase (FBA), heterologous transketolase (TKT), heterologous fructose/sedoheptulose biphosphatase (GLPX), heterologous transaldolase (TAL), heterologous ribose-5-phospate isomerase (RPI) and heterologous ribulose phosphate epimerase (RPE).
 7. The method of claim 1, wherein the non-naturally occurring microbe further expresses one or more heterologous cyclic formaldehyde dissimilation enzymes.
 8. The method of claim 7, wherein the expression of the one or more heterologous cyclic formaldehyde dissimilation enzymes is under the control of a formaldehyde responsive promoter.
 9. The method of claim 7, wherein the one or more heterologous cyclic formaldehyde dissimilation enzymes consist of heterologous glucose-6-phosphate isomerase (PGI), glucose-6-phosphate-1-dehydrogenase (ZWF), 6-phosphogluconolactonase (PGL), and 6-phosphogluconate dehydrogenase (GND).
 10. The method of claim 7, wherein the non-naturally occurring microbe contains a deletion of a phosphogluconate dehydratase gene (edd).
 11. The method of claim 1, wherein the non-naturally occurring microbe further expresses one or more heterologous CO₂ fixation pathway enzymes.
 12. The method of claim 11, wherein the expression of the one or more heterologous CO₂ fixation pathway enzymes is under the control of a formaldehyde responsive promoter.
 13. The method of claim 11, wherein the one or more heterologous CO₂ fixation pathway enzymes consist of heterologous carbonic anhydrase (CA), heterologous formate dehydrogenase (FDH), and heterologous formaldehyde dehydrogenase (FLD).
 14. The method of claim 1, wherein the methanol is the sole carbon source of the non-naturally occurring microbe. 